Linear amplification of specific nucleic acid sequences

ABSTRACT

This invention provides novel processes for amplifying nucleic acid sequences of interest, including linear and non-linear amplification. In linear amplification, a single initial primer or nucleic acid construct is utilized to carry out the amplification process. In non-linear amplification, a first initial primer or nucleic acid construct is employed with a subsequent initial primer or nucleic acid construct. In other non-linear amplification processes provided by this invention, a first initial primer or nucleic acid construct is deployed with a second initial primer or nucleic acid construct to amplify the specific nucleic acid sequence of interest and its complement that are provided. A singular primer or a singular nucleic acid construct capable of non-linear amplification can also be used to carry out non-linear amplification in accordance with this invention. Post-termination labeling process for nucleic acid sequencing is also disclosed in this invention that is based upon the detection of tagged molecules that are covalently bound to chemically reactive groups provided for chain terminators. A process for producing nucleic acid sequences having decreased thermodynamic stability to complementary sequences is also provided and achieved by this invention. Unique nucleic acid polymers are also disclosed and provided in addition to other novel compositions, kits and the like.

FIELD OF THE INVENTION

This invention relates to the field of recombinant nucleic acidtechnology, and more particularly, to processes for nucleic acidamplification, the post-termination labeling for nucleic acid sequencingand the production of nucleic acid having decreased thermodynamicstability.

All patents, patent applications, patent publications, scientificarticles, and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

The first system described for the successful in vitro exponentialamplification of target nucleic acids is the Polymerase Chain Reaction(PCR) (Saiki et al., 1985 Science 230; 1350-1354). PCR has been widelyused for allele determination, forensic identification, gene analysis,diagnostics, cloning, direct sequencing and other applications.Subsequently, Reverse Transcriptase (RT) was used to transform an RNAmolecule into a DNA copy allowing the use of RNA molecules as substratesfor PCR amplification by DNA polymerase. In addition, conditions havebeen described that allow certain DNA polymerases to perform reversetranscription by themselves (Myers, T. W. and Gelfand, D. H. [1991]Biochem. 30; 7661-7666), contents incorporated herein by refernce.Finally, Rose et al. (U.S. Pat. No. 5,508,178, also incorporated hereinby reference) have described the use of inverted repeat sequences aschoices for PCR primer sequences, allowing the use of a single primer toinitiate polymerization from each end of a target nucleic acid to createa PCR amplicon that in single-stranded form can be drawn as a“pan-handles” with self complementary sequences at each end. In order toutilize targets that lack inverted repeats, this group has alsodescribed various methods to introduce sequences into a PCR amplicon,such that the final product would have self-complementary sequences ateach end (U.S. Pat. Nos. 5,439,793, 5,595,891, and 5,612,199, each ofwhich is incorporated herein by reference).

Both the original PCR amplification system and various improved PCRsystems suffer from the limitation of a necessity for expensivededicated thermocyclers to provide the multiple temperature conditionsthat are intrinsic to the PCR method. This necessity is derived from theproblem that the extension of a primer creates a product that has astronger association with a template that the primer used to create it.As such, in a system like PCR, temperatures that allow binding of aprimer are too low to allow separation of the extended product from itstemplate and temperatures that are elevated enough to allow theseparation of the extended product are too high to allow another primingevent. The second priming event cannot take place until after the firstextended strand is separated from its template. As such, in PCRamplification, primer binding to template and the sequential release ofthe extended primers from the template have to be carried out atseparate distinct temperatures and require a thermocycler to providerepeated sequences of distinct thermal steps. The existence of discretecycles with different conditions also necessitates an optimization oftemperature for each individual temperature step as well as anappropriate timing for each step. Similar problems also apply whenligation is used in the LCR reaction (Backman, K. et al. European PatentPublication No. 0 320 308 B1, Landegren, U., et al., 1988 Science 241;1077, Wu, D. and Wallace, R. B. 1989 Genomics 4; 560, Barany, F. 1991Proc, Nat. Acad. Sci. USA 88; 189) where the temperature required forbinding individual probes is less than the temperature required torelease them after they have been stabilized by a ligation event. All ofthe foregoing documents are incorporated herein by reference.

Others have recognized these limitations and tried to overcome them byproviding means to accomplish multiple cycles under isothermalconditons. Examples of this are 3SR (Kwoh, D. Y. et al., (1989) Proc.Nat. Acad. Sci. USA 86; 1173-1177) and NASBA (Kievits., T. et al., 1991J. Virol. Methods 35; 273-286, the contents of each of which isincorporated herein by reference). Each of the preceding systems has thelimitation of a necessity for the introduction of an RNA promoter intothe structure of the nucleic acid being amplified. Consequently, thereis also a limitation that these systems are dependent upon a cyclingreaction between DNA and RNA forms of the sequence of interest. Adependency upon the production of an RNA intermediate introduces alimitation of susceptibility to RNases, enzymes that are ubiquitous inthe environment and are frequently present in biologically derivedspecimens. In addition, the nature of the design of these amplificationsystems has the further limitation that they require the presence offour distinct enzymatic activities: DNA polymerase, ReverseTranscriptase, RNase H and RNA polymerase. In the TMA reaction, theseactivities are provided by the Reverse Transcriptase and RNA polymeraseenzyme whereas in 3SR and NASBA they are provided by ReverseTranscriptase, RNase H and RNA polymerase enzymes. Each of theseactivities is required for the system to be functional, and as suchthere is a necessity for the manufacturer to test and titrate eachfunction individually, thereby increasing the cost compared to systemsthat utilize a single enzyme activity. In addition, at a minimum, atleast two different enzymes have to be used to provide all the necessaryfunctions, thus rendering these systems more expensive than those thatutilize a single enzyme. Furthermore, these systems requireribonucleotides as well as deoxyribonucleotides to be present asreagents for the reactions. The presence of multiple activities alsocreates more steps that are vulnerable to being inactivated by variousinhibitors that may be present in biological specimens.

In the Strand Displacement Amplification method described by Walker etal. (Proc. Nat. Acad. Sci. U.S.A. 1992, 89; 392-396, incorporated hereinby reference), isothermal amplification is carried out by the inclusionof a restriction enzyme site within primers such that digestion by arestriction enzyme allows a series of priming, extension anddisplacement reactions from a given template at a single temperature.However, their system has the limitation that besides the basicrequirement for a polymerase and substrates, three additional elementsare required in order to carry out their invention. First, there is anecessity for the presence of appropriate restriction enzyme sites atthe sites where priming is to take place; secondly, there is a necessityfor a second enzyme, a restriction enzyme, to be present, and lastlythere is a necessity for specially modified substrates, such as thioderivatives of dNTPs to be present. A variation of this method has beendescribed (U.S. Pat. No. 5,270,184, incorporated herein by reference)where the limitation of a necessity of a restriction enzyme site in thetarget has been eliminated by the use of a second set of primers thatare adjacent to the primers with the restriction enzyme sites. However,in this variation, a system is described that has a new limitation of arequirement for a second set of primers while retaining the other twolimitations of a need for a restriction enzyme and modified substrates.

Temperatures used for the various steps of full cycle amplification aredictated by the physical constraints that are intrinsic to each step. Assuch, in prior art the temperature used for complete displacement ofextended strands from templates is typically around 92-95° C. This hightemperature has been used to insure an adequate efficiency of separationsuch that an extended strand can be used as a template for subsequentreactions. When PCR was first described, the polymerase was derived fromE. coli and as such there was essentially complete thermal inactivationof the polymerase after each denaturation step that required theaddition of more enzyme (Saiki et al., 1985 230; 1350-1354; citedsupra). This problem was addressed by the use of a DNA polymerase from athermophlilic bacterium, T. aquaticus, in PCR reactions (Saiki et al.,1988 Science 239; 487-491). Each of the foregoing Saiki publications isincorporated herein by reference. Due to its inherent heat stability,enzyme was continuously present throughout the PCR cycles and no furtheradditions were required. Since that time, polymerases from otherthermophiles have also been isolated and used in full cycle reactions.However, even though they are more robust in their resistance to thermalinactivation, these polymerases all suffer from a limitation of having acertain level of inactivation after each denaturation step that isdictated by a half-life for that particular enzyme at the temperatureused for denaturation. Also the high denaturation temperature can alsodecrease the levels of dNTP substrates by hydrolysis and lead toinactivation of proteins that may be added to supplement the efficiencyor specificity of the reaction.

Full cycle PCR conditions have been modified such that lowerdenaturation temperatures could be used. Auer et al., (1996, Nucl. AcidsRes 24; 5021-5025, incorporated herein by reference) have described aprocedure that used dITP, a natural neutral-analogue of dGTP. By thissubstitution, they succeeded in avoiding amplification ofdouble-stranded DNA that may be present in their samples and onlyamplified RNA targets. By no means is there recognition or appreciationof a utility for DNA targets. In fact, they teach away since theirpurpose is to avoid the use of DNA targets as templates. Their teachingshave a limitation that the substitution dITP also necessitated acompensatory decrease in the temperatures used for the annealing (50°C.). In addition, the art described by Auer et al. relies upon the useof a nucleotide analogue that is known for a lack of discrimination forbase pairing, thereby introducing the possibility of random variationsbeing introduced into the sequence being amplified. When thesealterations are in the primer binding area they may cause problems inpriming efficiency and when they are in sequences between the primersthey may introduce difficulties in detection probes being able to bindefficiently. The present invention is capable of using bases thatexhibit normal levels of base pairing discrimination thereby avoidingthe mutagenic events that are part of the previous art.

Determination of the nucleic acid sequence of genes and genomes is amajor activity in both commercial and non-profit laboratories. The twobasic systems that have been employed for this purpose are the basespecific cleavage method described by Maxam and Gilbert (Proc. Nat.Acad. Sci. U.S.A. 1977, 74, 560-564) and the dideoxy method described bySanger et al. (Proc. Nat. Acad. Sci. U.S.A. 1977, 74, 5463-5467). Bothof the foregoing classical papers are incorporated herein by reference.Due to its ease of use the latter method is more commonly used. Both ofthese methods initially relied upon radioactive substrates for obtainingsequence information. For Maxam and Gilbert sequencing, this was mostcommonly carried out by end-labeling each strand and then separatingeach labeled end. For Sanger sequencing, either the primer is labeled orradioactive dNTP's are incorporated during strand extension. Sequencedata was produced by autoradiographic determination of the position ofradioactively labeled DNA bands of various lengths that had beenseparated by electrophoresis through a polyacrylamide gel.

In more recent years, sequencing methods have been improved by thesubstitution of non-radioactive labels. Non-radioactive labeling,potential positions for these labels and applications of their use aredisclosed by Engelhardt et al., in U.S. Pat. No. 5,241,060, which wasoriginally filed in 1982. Such labels can be in the oligo primer or inthe substrates used for synthesis, i.e. the dNTP or ddNTP nucleotides.Signal generating moieties can act directly as exemplified by the use offluorescently labeled primers (Beck et al., Nucleic Acids Res. 1989, 17;5115-5123) or indirectly as exemplified by the use of biotin labeledprimers (Ansorge et al., J. Biochem. Biophys. Methods 1986, 13; 315-323and). In addition, biotinylated nucleotides could be incorporated duringlimited primer extension (Sequenase Images™ Protocol Book 1993 UnitedStates Biochemical Corporation, Cleveland, Ohio). The foregoing fourdocuments are incorporated herein by reference. A limited extension isrequired to standardize the amount of band-shifting caused by themodification in the nucleotides.

However, primer labeling has the limitation that there can be secondarystructure or problematic sequences in the template strand that can causeinappropriate chain termination events that create ambiguities in theproper base assignment for that position. Incorporation of labeled dNTPsduring the extension of the primer also suffers from this limitation.This limitation is valid regardless of whether radioactive ornon-radioactive labels are used.

This limitation has been circumvented by the choice of the chainterminator nucleotide itself as the source of the label. This has beendescribed by Hobbs and Cocuzza in U.S. Pat. No. 5,047,519 and byMiddendorf et al., in U.S. Pat. No. 4,729,947 for fluorescently labeledddNTPs and by Middendorf et al., in U.S. Pat. No. 4,729,947 for biotinlabeled ddNTPs that were later marked by fluorescent avidin. (Forfurther reference refer to U.S. Pat. Nos. 5,027,880; 5,346,603;5,230,781; 5,360,523; and 5,171,534.). Each of the foregoing sevenpatents are incorporated by reference into this application. By thismethod, signals will be generated by strands that have incorporated achain terminator. The presence of strands that have been terminatedwithout the incorporated of a terminator nucleotide is now irrelevantsince they are incapable of signal generation. However, this method hasthe limitation that the presence of additional chemical groups thatprovide signal generation produce steric or other inhibitory problemsfor the polymerase directed incorporation of the labeled terminatornucleotide, thereby decreasing the efficiency of the reaction (Prober etal. in U.S. Pat. No. 5,332,666, incorporated herein). It has also beensuggested that biotinylated dideoxynucleotides could be used to providesignal generation, but these modified terminators were predicted toshare the same limitations as their fluoresceinated counterparts, i.e.difficulty in incorporation by most commonly used polymerases (S. Beck1990 Methods in Enzymology 184; 612-617, also incorporated herein). Somecompensation for this inefficiency of incorporation can be achieved byincreasing the amounts of polymerase in the reaction and/or byincreasing the amounts of template DNA being copied. These compensatorysteps suffer the limitation of increased costs associated with higheramounts of an expensive enzyme, DNA polymerase, or with preparation ofadequate amounts of high quality template

SUMMARY OF THE INVENTION

This invention provides for novel processes that are useful andapplicable in nucleic acid amplification, nucleic acid sequencing andthe production of unique nucleic acids having important properties, suchas decreased thermodynamic stability.

The present invention provides a process for linearly amplifying aspecific nucleic acid sequence. Initially, there are provided thespecific nucleic acid sequence of interest that is sought to beamplified, an initial primer or a nucleic acid construct comprising twosegments. The first segment (A) is unique, being characterized as (i)substantially complementary to a first portion of the specific nucleicacid sequence and (ii) capable of template-dependent first extension.The second segment (B) is uniquely characterized in the following fourrespects. First, it is (i) substantially non-identical to the firstsegment. Second, it is (ii) substantially identical to a second portionof the specific nucleic acid sequence. Third, the second segment (B) is(iii) capable of binding to a complementary sequence of the secondsegment. Fourth, the second segment (B) is (iv) capable of providing forsubsequent binding of a first segment of a second primer or nucleic acidconstruct to the first portion of the specific nucleic acid sequenceunder isostatic or limited cycling conditions. In this way, a secondprimer extension is produced and displaces a first primer extension.Also provided in this process are substrates, buffer and atemplate-dependent polymerizing enzyme. In carrying out thisamplification process, the specific nucleic acid sequence and the novelprimer or nucleic acid construct are incubated in the presence of thesubstrates, buffer and template-dependent polymerizing enzyme underisostatic or limited cycling conditions; thereby linearly amplifyingsaid specific nucleic acid sequence.

The present invention also provides a process for non-linearlyamplifying a specific nucleic acid sequence. In this process, there areprovided the specific nucleic acid sequence of interest sought to beamplified, a first initial primer or a nucleic acid construct for thespecific nucleic acid sequence of interest, a subsequent initial primeror a nucleic acid construct to the complement of the specific nucleicacid sequence of interest, and substrates, buffer and atemplate-dependent polymerizing enzyme. The first initial primer ornucleic acid construct comprises two segments. The first segment (A) isunique, characterized as being (i) substantially complementary to afirst portion of the specific nucleic acid sequence and (ii) capable oftemplate-dependent first extension. The second segment is also unique,being characterized with four features. First, it is (i) substantiallynon-identical to the first segment. Second, it is (ii) substantiallyidentical to a second portion of the specific nucleic acid sequence.Third, the second segment is (iii) capable of binding to a complementarysequence of the second segment. Fourth, the second segment is (iv)capable of providing for subsequent binding of a first segment of asecond primer or nucleic acid construct to the first portion of thespecific nucleic acid sequence under isostatic or limited cyclingconditions. In this way, a second primer extension is produced todisplace a first primer extension. The subsequent initial primer or anucleic acid construct to the complement of said specific nucleic acidsequence also comprises two segments. The first segment (A) ischaracterized as (i) being substantially complementary to a firstportion of the specific nucleic acid sequence and (ii) capable oftemplate-dependent first extension. The second segment (B) is uniquelycharacterized with four features. First, the second segment (B) (i)substantially non-identical to the first segment. Second, it is (ii)substantially identical to a second portion of the specific nucleic acidsequence. Third, the second segment (B) is (iii) capable of binding to acomplementary sequence of the second segment. Fourth, it is (iv) capableof providing for subsequent binding of a first segment of a subsequentprimer to the first portion of the specific nucleic acid sequence underisostatic or limited cycling conditions. Under such conditions and inthis way, a second primer extension is produced which displaces a firstprimer extension. To carry out this process, the specific nucleic acidsequence and the novel primer or nucleic acid construct are incubated inthe presence of the substrates, buffer and template-dependentpolymerizing enzyme under isostatic or limited cycling conditions;thereby non-linearly amplifying the specific nucleic acid sequence ofinterest.

Also provided by this invention is a process for non-linearly amplifyinga specific nucleic acid sequence. In this non-linear amplificationprocess, there are provided the specific nucleic acid sequence ofinterest sought to be amplified and its complement. Also provided is afirst initial primer or a nucleic acid construct for the specificnucleic acid sequence, this first initial primer or nucleic acidconstruct comprising two segments. The first segment (A) has two usefuland novel features. First, it is (i) substantially complementary to afirst portion of the specific nucleic acid sequence. Second, the firstsegment is (ii) capable of template-dependent first extension. Thesecond segment (B) has four useful and novel features. First, it is (i)substantially non-identical to the first segment. Second, the secondsegment (B) is (ii) substantially identical to a second portion of thespecific nucleic acid sequence. Third, it is (iii) capable of binding toa complementary sequence of the second segment. Fourth, the secondsegment (B) is (iv) capable of providing for subsequent binding of afirst segment of a subsequent first primer to the first portion of thespecific nucleic acid sequence under isostatic or limited cyclingconditions. Under such conditions and in this way, a second primerextension is produced which displaces the first primer extension. Alsoprovided in this process is a second initial primer or a nucleic acidconstruct complementary to the first primer extension. The secondinitial primer or nucleic acid construct typically comprises a singlesegment characterized by its being capable of template-dependentextension under isostatic or limited cycling conditions. Appropriatesubstrates, buffer and a template-dependent polymerizing enzyme are alsoprovided. To carry out this process of the present invention thespecific nucleic acid sequence and the novel primer or nucleic acidconstruct are incubated in the presence of the appropriate substrates,buffer and template-dependent polymerizing enzyme under isostatic orlimited cycling conditions. Under such incubation carried out underthose conditions, the specific nucleic acid sequence of interest isamplified non-linearly.

This invention further provides a process for non-linearly amplifying aspecific nucleic acid sequence of interest sought to be amplified. Inthis novel process, there are provided the specific nucleic acidsequence of interest, a singular primer or a singular nucleic acidconstruct capable of non-linear amplification and comprising threesegments. There is a first segment (a) that is (i) substantiallycomplementary to a first portion of the specific nucleic acid sequenceand is (ii) capable of template-dependent first extension. A secondsegment (b) is substantially identical to a second portion of thespecific nucleic acid sequence. The third segment (c) is substantiallyidentical to the first segment. The first primer extension is capable ofproducing sequences that are capable of hybridizing to said secondsegment and is also capable of self-priming and self-extending toproduce a complement to the third segment. Also provided araeappropriate substrates, buffer and a template-dependent polymerizingenzyme. In carrying out this amplifying process, the specific nucleicacid sequence and the primer or nucleic acid construct are incubatedtogether in the presence of the appropriate substrates, buffer andtemplate-dependent polymerizing enzyme. The specific nucleic acidsequence of interest is non-linearly amplified thereby.

Also provided by the invention at hand is a post-termination labelingprocess for nucleic acid sequencing. Here, the process comprises thefirst step of producing, in the presence of untagged or unlabeledsubstrates, untagged or unlabeled primer, polymerizing enzyme, bufferand an appropriate untagged or unlabeled terminator for each nucleotidebase, nucleic acid fragments corresponding to the nucleic acid sequenceof interest whose sequence is sought In this process, each of theterminators comprise a chemically reactive group that covalently bindsto a tagged molecule under conditions such that the internal sequencesare substantially non-reactive to the tagged molecules and the chemicalreactions do not substantially interfere with the separation of thefragments in a medium or matrix. After the production of fragments, thelatter are separated in a medium or matrix, followed by detection of theseparated fragments achieved by the detection of the tagged molecule inthe medium or matrix.

Another process provided by the present invention is a process forproducing nucleic acid sequences that have decreased thermodynamicstability to complementary sequences. In this process, at least onemodified nucleotide or nucleotide analog having a negatively chargedchemical moiety is incorporated into nucleic acid sequences which areproduced.

In addition to other aspects of this invention, there is provided asingle-stranded or double-stranded nucleic acid polymer selected fromthe group consisting of a linear nucleic acid, branched nucleic acid, aninverted nucleic acid and a peptide-nucleic acid, or a combination ofany of the foregoing. This nucleic acid polymer comprises at least onepurine or pyrimidine base comprising one negatively charged chemicalmoiety in one or both strands of the polymer.

All of these processes and polymers are described in greater detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts linear amplification by a novel primer.

FIG. 2 depicts non-linear amplification by a novel primer and a standardprimer.

FIG. 3 illustrates non-linear amplification by a pair of novel primers.

FIG. 4 shows non-linear amplification by a pair of novel primers thatcontain modifications that prevent part of their sequences from beingused as templates.

FIG. 5 depicts a series of reactions that can be carried out by anucleic acid construct with two 3′ ends where part of the construct iscapable of hairpin formation after template dependent extension.

FIG. 6 is a continuation of the process and events shown in FIG. 5.

FIG. 7 depicts a series of reactions that can be carried out by anucleic acid construct with two 3′ ends where each of the 3′ ends iscapable of hairpin formation after template dependent extension.

FIG. 8 is a continuation of the process and events shown in FIG. 7.

FIG. 9 illustrates template dependent extension and self priming/selfextension of a single primer capable of non-linear amplification.

FIG. 10 shows continuation of the process and events of FIG. 9.Potential intramolecular annealing and intermolecular annealing allowsthe continuous addition of sequences.

FIG. 11 are further illustrations of the modification of the processesand events in FIG. 10 wherein the initial primer contains a modificationthat does not allow a portion of the primer to be used as a template.

FIG. 12 are illustrations of a novel nucleic acid construct with two 3′ends that is capable of non-linear amplification.

FIG. 13 depict illustrations of another design for a novel nucleic acidconstruct with two 3′ ends that is capable of non-linear amplification.

FIG. 14 is a continuation of the processes and events shown in FIG. 13.

FIG. 15 depict illustrations of another design for a novel nucleic acidconstruct with two 3′ ends that is capable of non-linear amplification.

FIG. 16 is a continuation of the processes and events shown in FIG. 15.

FIG. 17 shows gel assays of isothermal amplifications of a targetcreated by PCR.

FIG. 18 are results of a gel assay and a plate assay for isothermalamplification of HPV plasmid DNA.

FIG. 19 shows the results of a gel assay for PCR reactions with carboxydUTP and normal dTTP under various reaction conditions defined therein.

FIG. 20 summarizes the results of FIG. 19.

FIG. 21 shows the effects of various levels of MgCl₂ on PCR synthesis inthe presence of carboxy dUTP.

FIG. 22 are the results of a gel assay for the ability of variouspolymerases to carry out PCR synthesis in the presence of carboxy dUTP.

FIG. 23 shows the effects of various levels of MgCl₂ on PCR synthesis inthe presence of carboxy dUTP with various enzymes.

FIG. 24 shows the effects of various levels of MgCl₂ on PCR synthesis inthe presence of carboxy dUTP and PCR Enhancer with various enzymes.

FIG. 25 shows the effects of various additives on PCR synthesis in thepresence of carboxy dUTP.

FIG. 26 shows the sequences for the template (SEQ ID NO: 13-18) andprimers (SEQ ID NO: 11-12) used for PCR synthesis in the presence ofcarboxy dUTP.

FIG. 27 are the results of a gel assay for various combinations ofprimers for PCR synthesis in the presence of carboxy dUTP.

FIG. 28 are the results of a gel assay for various combinations ofprimers for PCR synthesis in the presence of carboxy dUTP at differenttemperatures shown therein.

FIG. 29 are the results of gel assays for various conditions used forpost-synthetic attachment of a fluorescent marker.

FIG. 30 is a negative image of the results of FIG. 29.

The definitions below are useful to an understanding of the presentinvention and this disclosure.

Definitions

Isostatic conditions refer to substantially constant temperature and/orchemical conditions.

Limited cycle conditions refer to a series of temperatures where thehighest temperature used is below the temperature required forseparation of an extended primer from its template.

Full cycle conditions refer to a series of temperatures where at leastone temperature is used that is sufficient for separation of an extendedprimer from its template.

Linear amplification is carried out when two or more copies of only onestrand of nucleic acid are produced.

Non-linear amplification is carried out when two or more copies of anucleic acid sequence are produced from each strand of a nucleic acidand its complement.

An initial primer is a primer or primer construct that has not beenextended.

A standard primer is a primer that is not substantially involved insecondary structure formation with sequences synthesized afterextension.

Extended sequences are sequences synthesized in a template dependentmanner which are substantially neither identical or complementary to anysequences in primers or primer constructs.

A segment of a nucleic acid is substantially identical to anothersegment when the complement of the said other segment is capable ofacting as a template for extension of the said first segment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods of use for novelprimers and nucleic acid constructs that a) contain at least one segmentthat has self-complementary sequences or is capable of forming asecondary structure after template-dependent extension and b) arecapable of producing two or more copies of a specific nucleic acidsequence under appropriate conditions in the presence of an appropriatespecific template under appropriate conditions.

All methods of target amplification that use primer binding andextension reactions for synthesis of a specific nucleic acid sequencehave the necessity to regenerate a binding site or sites or tosynthesize a new primer binding site or sites if two or more copies ofthis sequence are desired. In all methods of art that have beenpreviously described, outside modulating factors have been used toregenerate or create primer binding sites. These factors have includedthermal denaturation as exemplified by PCR, endonucleases as exemplifiedby 3SR, and restriction enzymes and modified nucleotides as exemplifiedby SDA.

In certain aspects of the present invention, novel primers and nucleicacid constructs are disclosed that have as an inherent characteristicthat at least one segment of a novel primer or nucleic acid construct iscapable of secondary structure formation under appropriate conditions.In the present invention, the formation of a secondary structure canprovide for regeneration of binding sites such that they can be used formultiple binding and extension of novel primers or nucleic acidconstructs without the necessity for any of the outside modulatingfactors described above.

In previous art there has been a necessity for the presence of a primerbinding site in each complementary strand of a target nucleic acid inorder to achieve non-linear amplification. In certain aspects of thepresent invention, the formation of secondary structures overcomes thislimitation such that a single primer can be used that is complementaryto only one nucleic acid strand and not the other, but yet is stillcapable of carrying out non-linear amplification of a desirable nucleicacid sequences.

The novel primer and nucleic acid constructs of the present inventionare capable of use in linear and non-linear amplification systems thatrequire only a single primer or more than one primer under isostatic,limited cycle or full cycle conditions. The capability for formation ofsecondary structures is due to the presence of self-complementarysequences in a novel primer or nucleic acid construct or it may bederived from the template dependent incorporation of sequences that arecomplementary to a segment of the novel primer or nucleic acidconstruct. It may also be derived from both pre-existing andpost-synthesis sequences. The novel primer and nucleic acid constructsof the present invention can be linear molecules with a single polarity,constructs with more than one polarity or branched nucleic acids.Methods of synthesis and examples of use of such constructs havepreviously been disclosed in (U.S. patent application Ser. No.08/749,266; U.S. Pat. No. 5,462,854, both documents incorporatedherein). In certain aspects of the present invention, the novel primerand nucleic acid constructs comprise at least two segments: a firstsegment that is capable of binding to a template and using it forextension and a second segment that is substantially identical tosequences of the target of interest such that extension of the firstsegment allows formation of a secondary structure formed byself-hybridization of the second segment with the extended sequences. Incertain aspects of the present invention, the novel primer and nucleicacid constructs comprise at least three segments: the first and secondsegments being defined as above and a third segment which is capable ofacting as an intrastrand or intraconstruct template for self-extension.

Segments can be joined together either covalently or non-covalently.Means of joining segments through covalent linkages can include but arenot limited to the phosphate backbone of normal linear nucleic acids,constructs that have more than one polarity and branched DNA constructs.Methods for synthesizing these constructs have been described in U.S.patent application Ser. No. 08/749,266, filed on Nov. 15, 1996, contentsof which are incorporated herein. Means of joining segments bynon-covalent linkages can include but are not limited to ligand-receptorbonds and complementary base pairing. The segments may be adjacent toeach other or they may be spatially separate from each other. Thesequences of the segments may be distinct from each other or they may besubstantially or partially complementary or identical to each other.

The formation of useful secondary structures can be augmented byadditional elements in the design of the novel primers and nucleic acidconstructs of the present invention. For instance, secondary structurescan be introduced into the sequences of the novel primers of the presentinvention that can allow extension-dependent secondary structures toform more easily. Supplementary elements can also be included in thereaction mixture to favor the formation of appropriate secondarystructures. These elements can include but are not limited to proteinssuch as single-stranded binding protein, the T4 gene 32 protein, Rec Aprotein and various helicases. These elements can also include but arenot limited to chemical reagents such as Formamide or DMSO. Theseelements can also include but are not limited to modified nucleotidesthat either raise or lower the Tm of a nucleic acid sequence. Themodified nucleotides can pre-exist in the novel primers and nucleicacids constructs, they can be incorporated during the extensionreactions or they can be both.

The various novel primers and novel nucleic acid constructs of thepresent invention overcome many of the limitations of previous systems.In contrast to methods that have been previously described in the artthat depend upon the use of a thermocycler, certain aspects of thepresent invention have no necessity for a strand separation event priorto a new priming event. Additionally, the present invention has norequirements for multiple enzyme arrangements, ribonucleotides or thepresence of a promoter sequence as are intrinsic to isothermal systemsthat are dependent on the generation of an RNA intermediate such as 3SR,NASBA and TMA. Nor is there a requirement for esoteric modified reagentsand a supplementary restriction enzyme as has been described for theisothermal SDA system.

Also included in the present invention are novel methods andcompositions that can be used for labeling of nucleic acids. These canbe used in conjunction with various aspects of the present invention ormay be used in conjunction with methods described in previous art.

This invention provides for a process to amplify linearly a specificnucleic acid sequence of interest that one seeks to amplify. Such aprocess includes the step of providing the following components andreagents: the specific nucleic acid sequence of interest, an initialprimer or a nucleic acid construct comprising two segments, andappropriate substrates, buffer and a template-dependent polymerizingenzyme. The two segments of the initial primer or nucleic acid constructinclude (A) a first segment having two defined characteristics. First,it is (i) substantially complementary to a first portion of the specificnucleic acid sequence and second, it is (ii) capable oftemplate-dependent first extension. The second segment (B) has fourdefined characteristics. First, the second segment (B) is (i)substantially non-identical to the first segment. Next, it is (ii)substantially identical to a second portion of the specific nucleic acidsequence. Third, the second segment (B) is (iii) capable of binding to acomplementary sequence of the second segment. Fourth, this secondsegment is (iv) capable of providing for subsequent binding of a firstsegment of a second primer or nucleic acid construct to the firstportion of the specific nucleic acid sequence under isostatic or limitedcycling conditions. In so doing, a second primer extension is producedand that displaces a first primer extension. Another important step ofthis linear amplification process is that of incubating the specificnucleic acid sequence and the novel primer or nucleic acid construct inthe presence of the appropriate substrates, buffer andtemplate-dependent polymerizing enzyme under isostatic or limitedcycling conditions; thereby linearly amplifying the specific nucleicacid sequence of interest that one seeks to amplify.

In other aspects of the just-described process, the initial primer ornucleic acid construct and the second primer or nucleic acid constructcan be the same, or they can be different. Furthermore, at least onemodified nucleotide or nucleotide analog can be usefully incorporatedinto various components or elements of the process, including the firstsegment, the second segment, or the primer extension product, or any ofthe foregoing elements for that matter. Such a modified nucleotide ornucleotide analog can be usefully incorporated into the second segmentdefined above. When usefully incorporated into the second segment, sucha modified nucleotide or nucleotide analog increases the thermodynamicstability of the first segment to its complement in the primerextension. The modified nucleotide or nucleotide analog can comprise anintercalating agent, for example.

Those skilled in the art will appreciate that the first segment or theprimer extension product, both of these elements, can comprise at leastone modified nucleotide or nucleotide analog. In such instances, themodified nucleotide or nucleotide analog decreases the thermodynamicstability of the first segment or the primer extension product to itscomplement. Such thermodynamic stability decreasing modified nucleotidesor nucleotide analogs comprise, for example, a negatively chargedchemical group, such as a carboxylic acid.

With respect to the nucleic acid form, the initial primer or nucleicacid construct or the second primer or nucleic acid construct (or bothprimers and nucleic acid constructs), can comprise a number of nucleicacids. These include but are not limited to linear nucleic acid,branched nucleic acid, an inverted nucleic acid and a peptide-nucleicacid, or a combination of any of the preceding. Further description oflinear amplification follows immediately below.

Linear Amplification with One Stem-loop Forming Primer

In one aspect of the present invention, linear amplification of aspecific nucleic acid sequence is carried out under isostatic or limitedcycle conditions by the use of a single novel primer or a single novelnucleic acid construct that has at least two segments. The novel nucleicacid constructs of the present invention can have more than one polarityor they can be branched DNA. Methods for synthesizing these constructshave been described in U.S. patent application Ser. No. 08/749,266,cited supra and incorporated herein. The first segment of a novel primeror nucleic acid construct comprises sequences that are substantiallycomplementary to sequences that are present in a target nucleic acidsequence. The second segment of a novel primer or nucleic acid constructcomprises sequences that are substantially identical to sequences thatare present in the target nucleic acid. A novel nucleic acid constructcan have one or more copies of the first and second segments. Templatedependent extension of the novel primer or nucleic acid construct cancreate a product that has a stem-loop structure formed byself-hybridization as well as extended sequences that are not identicalto or complementary to sequences that comprise the novel primer ornucleic acid construct.

This product can be formed by a continuous series of the following stepsthat are illustrated in FIG. 1. Template dependent extension of a novelprimer or nucleic acid construct produces in the extended portionsequences that are complementary to sequences that comprise the secondsegment of the said novel primer or nucleic acid construct. Theseself-complementary regions can remain bound to the template or can formself-complementary structures. The formation of a secondary structurecan provide for removal of all or part of the first segment of theextended novel primer from the template. This would allow anotherinitial primer to bind to template sequences prior to removal of thefirst extended novel primer from the template. Extension of the secondprimer on the template can lead to displacement of the first extendedprimer from the template. This is in contrast to previous art whereseparation of an extended primer always takes place prior to use of thetemplate for another binding and extension reaction. By these means, asingle template can provide for two or more initiation events underisostatic conditions. In addition, this method can be used under limitedcycling conditions where all temperatures are below those of the Tm ofan extended product and its template. In a continuing process, formationof a secondary structure in the second extended novel primer can providefor binding and subsequent extension of a third novel primer. In thisway, in the absence of denaturing conditions, the novel process of thepresent invention can provide for multiple priming, extension andrelease events from a single strand of a nucleic acid template.Furthermore, all of these steps can take place simultaneously andcontinuously under isostatic conditions.

A novel nucleic acid construct with multiple identical first and secondsegments could also be used to carry out linear amplification by thesame processes that have been depicted in FIG. 1. This novel constructcould potentially enjoy an increase in efficiency compared to a linearconstruct with single polarity. The binding and extension of one of thefirst segments of a construct molecule results in a localized highconcentration of other first segments of the construct that can bind tothe regenerated primer binding site. After multiple priming andextension reactions, a construct can be created that comprises multiplecopies of a single strand of target DNA.

The ability of extended novel primer and nucleic acid constructs to formself-complementary structures can be realized under appropriateconditions. Previous art has indicated that the association anddissociation of short complementary oligonucleotides occurs as anequilibrium reaction whose characteristics are determined by thetemperature, salt conditions, base content and length of thecomplementary sequences. The influence of these factors has beenreviewed by J. G. Wetmur ([1991] Crit. Rev. Biochem. Mol. Biol. 26;227-259, incorporated herein). Although larger strands of complementaryDNA exist as double-stranded molecules in stable configurations that donot readily dissociate over a wide range of conditions, it is well knownthat they do form temporary and localized relaxations of interstrandbonding. The term “breathing” has been used to describe this localizeddisruption of hydrogen bonding. A pathway for “breathing” to createtwo-dimensional structures in double-stranded DNA molecules that containpalindromic sequences has been described by A. Kornberg and T. A. Bakerin “DNA Replication, 2^(nd) Edition” (1992) W. H. Freeman and Co. NY,N.Y., pages 44-46; the contents of which are incorporated herein byreference.

In the present invention, as described above, the transition of asegment of a linear double-stranded molecule to an intra-strandstem-loop structure can allow primer initiation events to take placeprior to separation of an extended primer from its template. Theequilibrium between these two structures is dependent upon a number offactors. First, for successful primer binding, the segment of theinitial primer that binds to the target must be of appropriate lengthand base composition so as to allow stable priming at the temperaturebeing used for the reaction. Second, the segment of the primer thatparticipates in self-hybridization after an extension of the initialprimer must be of appropriate length and base composition such that apartial dissociation of the extended primer from the template can allowthe formation of a sufficiently stable secondary structure, i.e., thestem of a stem-loop structure.

Temperatures appropriate for these reactions are below those that wouldbe required for separation of an extended primer from its template. Inan isostatic reaction, a single temperature can be used for binding,extension and secondary structure formation. Or if so desired, limitedcycling conditions can be used where different temperatures are used tooptimize these events. The use of different temperatures for limitedcycling may be useful for primer binding, primer extension or alocalized separation of some of an extended product from its template.The temperatures being used for any and all of these steps should alsobe appropriate for the particular polymerase being used in thereactions.

Intra-molecular complementary regions in an extended primer have beenutilized previously by Rose et al. (U.S. Pat. Nos. 5,595,891, 5,612,199,both incorporated herein) to provide identical binding sites on eachstrand of a target nucleic acid in order to allow the use of a singleprimer for PCR amplification. However, all examples and teachingsprovided by Rose et al. require heating steps to separate an extendedprimer from its template prior to use of the template for the nextprimer binding and extension events, i.e. multiple cycles of completedenaturation in a thermocycler. Studies with single-stranded RNA haveshown that as the size of the loop increases there is a diminishedchance for intrastrand stem formation (R. L P. Adams et al., in “TheBiochemistry of the Nucleic Acids” [1992] Chapman & Hall, London, U.K.,incorporated herein). Yet, the methods provided by Rose et al. for PCRamplification with either natural or artificially introduced invertedrepeat sequences as primer sites utilize a preferred separation of100-2,000 nucleotides and more preferably of 500-10,000 bases betweencomplementary sequences that form the stem of a stem-loop structure.Such a direction teaches away from the methods and compositionsdisclosed in the present invention where complementary sequences aresufficiently proximate to each other that formation of a stem-loopstructure could facilitate the removal or partial removal of the firstsegment of an extended primer from its template to regenerate a bindingsite without the necessity of imposition of conditions that wouldprovide for the complete separation of an extended primer from itstemplate. In addition, the teachings provided by Rose et al. precludethe use of self-complementarity in the primer as a means of allowingamplification under isothermal or limited cycle conditions since theiroperating range would make secondary structure formation energeticallyunfavorable under isothermal or limited cycle conditions. The gain inenergy created by formation of a stable stem structure is compromisedand outweighed by the energy cost of displacing a long strand from itscomplement to form the loop portion of a stem-loop structure. Thus fullcycle conditions are required to regenerate a primer binding site. Theconsequences of the teachings and processes of Rose et al. lead toproducts in which the extended sequences are always in the loop of astem-loop structure whereas in this aspect of the present invention, theproduct of the novel primers and processes have the extended sequencesessentially outside of potential stem-loop structures.

The aspect of the invention that has been described above findsparticular utility in the preparation of labeled single-stranded DNAprobes and for determining the sequences of nucleic acids. Prior to thedisclosure of the present invention, the most commonly used methods forobtaining single-stranded DNA probes have been dependent upon multiplestrand denaturation events provided by a thermocycler, or by the use ofRNA polymerase with templates that contain an RNA promoter. Processesthat depend upon multiple strand denaturation events suffer from alimitation of loss of a certain amount of reagents and enzyme activityat the high temperatures required for denaturation of templates. Eventhermostable polymerases are not completely immune to the effects ofdenaturation condition temperatures and have various half-lives at thesetemperatures. Also the use of such conditions precludes the use of someenzymes that are completely inactivated by such temperatures. Theseprocesses also have the limitations of the need of a thermocycler.Processes that are dependent upon production of RNA suffer from thelimitations associated with a need for introducing an RNA promoter intoassociation with the sequences desired for a probe and from limitationsthat are intrinsic to a product that is more labile than DNA. Themethods disclosed for the use of isostatic or limited cycle conditionsin the present invention can be used with or without a thermocycler.They allow the use of a wider array of enzymes, reagents are notsubjected to extreme destabilizing conditions and stable reusable DNAprobes are the final product.

This aspect of the present invention can also be used in sequencing byallowing a template to be used a multiple number of times underisostatic or limited cycle conditions. Previous art has only been ableto accomplish this by the use of multiple strand denaturation events ina thermocycler. The limitations cited previously for multiple stranddenaturation events are also applicable to this use. In addition, thereis an additional limitation that the high temperatures required fordenaturation can contribute to heat-induced depurination or deaminationevents that can create sequence ambiguities. Application of the methodsof the present invention for multiple rounds of sequencing from atemplate offers the advantages of independence from the necessity of athermocycler, utility of a wider array of enzymes and moderation ofthermal effects upon the integrity of templates and reagents.

The present invention also provides a process for non-linearlyamplifying a specific nucleic acid sequence. Non-linear amplificationcomprises a first step of providing the following components orreagents: the specific nucleic acid sequence of interest sought to beamplified, a first initial primer or a nucleic acid construct for thespecific nucleic acid sequence, a subsequent initial primer or a nucleicacid construct to the complement of said specific nucleic acid sequence,and appropriate substrates, buffer and a template-dependent polymerizingenzyme. The just-described first initial primer or nucleic acidconstruct comprises two segments. First, there is a first segment (A)that has two defined characteristics. It is (i) substantiallycomplementary to a first portion of said specific nucleic acid sequenceand it is (ii) capable of template-dependent first extension. The secondsegment of the first initial primer has four defined characteristics.First, it is (i) substantially non-identical to the first segment (A).Second, it is (ii) substantially identical to a second portion of thespecific nucleic acid sequence. A third characteristic of the secondsegment is its capability (iii) for binding to a complementary sequenceof the second segment. A fourth characteristic of the second segment isits (iv) capability for providing for subsequent binding of a firstsegment of a second primer or nucleic acid construct to the firstportion of the specific nucleic acid sequence under isostatic or limitedcycling conditions. Under such conditions, a second primer extension isproduced to displace a first primer extension.

With respect to the subsequent initial primer or nucleic acid construct,this element comprises two segments, a first segment (A) and a secondsegment (B). The first segment (A) is (i) substantially complementary toa first portion of the specific nucleic acid sequence and it is (ii)capable of template-dependent first extension. Four characteristicsdefine the second segment (B). First, the second segment (B) is (i)substantially non-identical to the first segment. Second, it is (ii)substantially identical to a second portion of the specific nucleic acidsequence. Third, the second segment (B) is (iii) capable of binding to acomplementary sequence of the second segment. The fourth characteristicof the second segment (B) is (iv) its capability for providingsubsequent binding of a first segment of a subsequent primer to thefirst portion of the specific nucleic acid sequence under isostatic orlimited cycling conditions. Under such conditions, a second primerextension is produced and that displaces a first primer extension. Thesecond step of this process includes incubating the specific nucleicacid sequence and the novel primer or nucleic acid construct in thepresence of the appropriate substrates, buffer and template-dependentpolymerizing enzyme under isostatic or limited cycling conditions. Thespecific nucleic acid sequence of interest is thereby amplifiednon-linearly thereby.

In the just-described non-linear amplification process, the firstinitial primer or nucleic acid construct and the second initial primeror nucleic acid construct can be the same, or they can be different.Modified nucleotides or nucleotide analogs can be usefully incorporatedas additional elements. For example, these can be incorporated into thefirst segment or the second segment of the first initial primer ornucleic acid construct, or into the first segment or the second segmentof the second initial primer or nucleic acid construct. Or, modifiednucleotides or nucleotide analogs can be incorporated into any primerextension products. For that matter, modified nucleotides or nucleotideanalogs can be incorporated into or used to modify any of the precedingelements.

In further embodiments of this non-linear amplification processjust-described above, the second segment of the first initial primer orthe second initial primer can comprise a modified nucleotide ornucleotide analog which serves to increase the thermodynamic stabilityof the first segment to its complement in the primer extension product.Such modified nucleotides or nucleotide analogs comprise or take theform of, for example, an intercalating agent.

In other aspects of the process at hand, the first segment of the firstinitial primer or the first segment of the second initial primer (orboth), or even the primer extension product (or any combination of thepreceding elements, for that matter) can comprise a modified nucleotideor nucleotide analog. Here, the modified nucleotide or nucleotide analogserves to decrease the thermodynamic stability of the first segment orthe primer extension or both, to their corresponding complement. Suchstability decreasing modified nucleotides or nucleotide analogs cancomprise negatively charged chemical groups, such as carboxylic acid.

Another aspect of the just-describe non-linear amplification process isthe type or form of nucleic acid. Here, the first initial primer ornucleic acid construct, or the second initial primer or nucleic acidconstruct, or both, comprises any number or form of nucleic acids. Suchmembers include but are not limited to linear nucleic acid, branchednucleic acid, an inverted nucleic acid and a peptide-nucleic acid, orcombinations of any of the foregoing.

Another significant non-linear amplification process is provided by thepresent invention. This process amplifies non-linearly a specificnucleic sequence and comprise a first step of providing the followingcomponents and reagents: the specific nucleic acid sequence and itscomplement: a first initial primer or a nucleic acid construct for thespecific nucleic acid sequence, a second initial primer or a nucleicacid construct complementary to said first primer extension, andappropriate substrates, buffer and a template-dependent polymerizingenzyme. The first initial primer or nucleic acid construct comprises twosegments: a first segment (A) and a second segment (B). With respect tothe former, two characteristics define it. First, it is (i)substantially complementary to a first portion of the specific nucleicacid sequence and second, it is (ii) capable of template-dependent firstextension. With respect to the second segment (B), four characteristicsdefine this element. First, it is (i) substantially non-identical to thefirst segment. Second, it is (ii) substantially identical to a secondportion of the specific nucleic acid sequence. The third characteristicof the second segment (B) is its (iii) capability for binding to acomplementary sequence of the second segment. A fourth characteristic ofthe second segment (B) is (iv) its capability for providing subsequentbinding of a first segment of a subsequent first primer to the firstportion of the specific nucleic acid sequence under isostatic or limitedcycling conditions. Under such conditions, a second primer extension isproduced and that displaces the first primer extension. The secondinitial primer or nucleic acid construct comprises a segmentcharacterized by its capability for template-dependent extension underisostatic or limited cycling conditions. The important step of thisprocess is, of course, that of incubating the specific nucleic acidsequence and the novel primer or nucleic acid construct in the presenceof the appropriate substrates, buffer and template-dependentpolymerizing enzyme under isostatic or limited cycling conditions. Thespecific nucleic acid sequence of interest is amplified non-linearlythereby.

Other aspects or features can be incorporated into the last-describedprocess for non-linear amplification. One important feature is theinclusion of modified nucleotides or nucleotide analogs. For example, atleast one modified nucleotide or nucleotide analog can be incorporatedor used to modify any of the following member elements in the process:the first segment or the second segment of the first initial primer ornucleic acid construct, the segment of the second initial primer ornucleic acid construct, the primer extension, or any of the foregoing orcombinations of any of the foregoing. Equally significant is theinclusion of at least one modified nucleotide or nucleotide analog intothe second segment of the first initial primer. The inclusion of suchmodified nucleotides or nucleotide analogs serves to increase thethermodynamic stability of the first segment to its complement in theprimer extension. Modified nucleotides or nucleotide analogs are wellknown in the art, and include, for example, intercalating agents.

Furthermore, the first segment of the first initial primer or thesegment of the second initial primer (or both), or their primerextension (or for that matter, any combinations of the foregoing) can bemodifed or incorporated with at least one modified nucleotide ornucleotide analog. Such modified nucleotides or nucleotide analogs serveto decrease the thermodynamic stability of the first segment or theprimer extension (or both) to their respective complements. Modifiednucleotides or nucleotide analogs that serve to decrease stability cancomprise a negatively charged chemical group, such as carboxylic acid.

As in the case of other processes for non-linear amplification describedin this application, the form or type of nucleic acid can vary. Thefirst initial primer or nucleic acid construct, or the second initialprimer or nucleic acid construct, or both, can comprise nucleic acidselected from any of the following: linear nucleic acid, branchednucleic acid, inverted nucleic acid and peptide-nucleic acid (orcombinations of any of the foregoing).

The invention at hand also provides another process for the non-linearamplification of a specific nucleic acid sequence of interest sought tobe amplified. This process comprises the first step of providing thefollowing components and reagents: the specific nucleic acid sequence ofinterest; a singular primer or a singular nucleic acid construct capableof non-linear amplification, and appropriate substrates, buffer and atemplate-dependent polymerizing enzyme. The singular primer or nucleicacid construct comprises three segments, (a), (b) and (c). The firstsegment (a) is (i) substantially complementary to a first portion of thespecific nucleic acid sequence and (ii) is capable of template-dependentfirst extension. The second segment (b) is substantially identical to asecond portion of the specific nucleic acid sequence. The third segment(c) is substantially identical to the first segment. The first primerextension is capable of producing sequences that are capable ofhybridizing to the second segment and is also characterized by itscapability for self-priming and self-extension to produce a complementto the third segment. Following the first step of this process, thespecific nucleic acid sequence and the primer or nucleic acid constructare incubated in the presence of the appropriate substrates, buffer andtemplate-dependent polymerizing enzyme. After incubation; the specificnucleic acid sequence is amplified non-linearly thereby.

Other embodiments for the last-described process for non-linearamplification are provided by the present invention. For example, theprocess can be carried out under conditions selected from isostaticconditions, limited cycling conditions and full cycling conditions.

In addition, modified nucleotides or nucleotide analogs can be used inthe modification of various elements of the process. For example, any orall of the first segment, the second segment, the third segment, thefirst primer extension, the second primer extension, can include orcomprise at least one modified nucleotide or nucleotide analog.Furthermore, modified nucleotides or nucleotide analogs can beincorporated into any or all of the first segment, the second segment,the third segment, the first primer extension and the self primingextension.

Those skilled in this art will also appreciate that the singular primeror nucleic acid construct can comprise a number of nucleic acid forms,including, for example, linear nucleic acid, branched nucleic acid,inverted nucleic acid and peptide-nucleic acid, or combinations of anyof the foregoing. Skilled artisan will further appreciate that firstprimer extension can be carried out under various conditions, includingfor example, limited substrate conditions, limited extension duration,or both.

With respect to any of the processes described above for amplificationof specific nucleic acid sequences of interest, be it linear ornon-linear ampification, the specific nucleic acid sequence can be insingle-stranded or double-stranded form. Moreover, the specific nucleicacid sequence can be found or is contained in a fragment. Such afragment can be produced by a number of means, including physical means(sonication, heat, or both), chemical means (acid treatment),physico-chemical means and enzymatic means (nucleases, e.g.,endonucleases, and restriction enzymes).

Non-linear amplification is further described below.

Non-Linear Amplification with Stem-loop Forming Primers and Constructs

Non-linear amplification of a desired sequence can be carried out whenbinding sites on each strand are used by primers or nucleic acidconstructs. In another aspect of the present invention, non-linearamplification can be carried out under isostatic or limited cycleconditions when at least one of the said primers or constructs is anovel primer or construct with a first and second segment. The novelnucleic acid constructs of the present invention can have more than onepolarity or they could be branched DNA. Methods for synthesizing theseconstructs have been described in U.S. patent application Ser. No.08/749,266, cited supra and incorporated herein. The first and secondsegments are as defined previously. The first segment of a novel primeror nucleic acid construct comprises sequences that are substantiallycomplementary to sequences that are present in a target nucleic acidsequence. The second segment of a novel primer or nucleic acid constructcomprises sequences that are substantially identical to sequences thatare present in the target nucleic acid.

When primers are used for non-linear amplification, the binding site onone strand is used by a novel primer with a first and second segment andthe binding site on the other strand may be used by either a standardprimer or another novel primer. A single novel primer may be used byitself when the binding sites in each strand are substantially similarto each other. When constructs are used for non-linear amplification,the construct is a novel construct that comprises one or more firstsegments that are complementary to one strand and one or more firstsegments that are complementary to the other strand of the targetnucleic acid. The construct also comprises one or more second segmentsthat are identical to one strand and can also comprise one or moresecond segments that are identical to sequences in the other strand. Thefirst segments of the novel construct may be substantially identical toeach other or they may be substantially dissimilar to each other. Thesecond segments of the novel construct may be substantially identical toeach other or they may be substantially dissimilar to each other. It isalso understood that combinations of standard primers, novel primers,constructs and novel constructs may also be used together as long as atleast one of them contains a first and second segment.

As described previously, the binding and extension of a novel primer ornucleic acid construct can allow the use of a template for multipleprimer binding and extension events under isostatic or limited cycleconditions. As new binding and extension events occur, they allow theseparation of the nucleic acid strand that had previously been extendedon that template. This results in the production of single-strandednucleic acid strands that can be used as templates for binding of asecond primer or nucleic acid construct without a necessity for adenaturation event since they are already in single-stranded form. Whenone primer is a standard primer and the other is a novel primer, thefinal product of template dependent bindings and extensions can be adouble-stranded molecule that on one end comprises a stem-loop structureon each strand. When both primers are novel primers, the final productof template dependent bindings and extensions can be a double-strandedmolecule that on each end comprises a stem-loop structure on eachstrand. When a construct comprises two first segments, each of which iscomplementary to one strand or the other and one second segment that iscomplementary to only one strand, the final product can be a singlemolecule that has complementary stem-loop structures. When a constructcomprises two first segments, each of which is complementary to onestrand or the other and two second segments, each of which is identicalto one strand or the other, the final product can be a single moleculethat has two pairs of complementary stem-loop structures.

A non-linear amplification product can be synthesized by a novel primerand a standard primer by a continuous series of the following stepsunder isostatic or limited cycle conditions. The novel primer binds to atarget strand and there is the same series of extension, secondarystructure formation, regeneration of a primer binding site, secondbinding, second extension and separation of the first extended primerfrom the template as described previously for linear amplification witha single novel primer. As extended novel primers are displaced bycontinuous binding and extension of other novel primers, thesesingle-stranded products can bind standard primers and allow them to beextended to create a full double-stranded amplicon. This potentialseries of events is depicted in FIG. 2. The resulting double strandedstructure contains in each strand self-complementary sequences thatflank, in one strand, a sequence complementary to the primer bindingsite for the novel primer, and in the other strand a sequence identicalto the primer binding site for the novel primer. As a result of this,each strand is capable of forming a stem-loop structure at one end ofthe amplicon. The exposure of the primer binding site in thesingle-stranded loop structure can then generate a further series ofprimer binding and displacement reactions by the same process previouslydepicted in FIG. 1, thereby allowing generation of non-linearamplification of the sequences of interest under isostatic or limitedcycle conditions. This product is different than that created by Rose etal. by non-linear amplification since their processes led to theextended sequences always being located between self-complementaryregions whereas in this aspect of the present invention, the extendedsequences are outside of the stem-loop regions. In addition, theprocesses of this aspect of the present invention regenerate a bindingsite by secondary structure formation whereas in Rose et al., thebinding site is in the stem region of a potential stem-loop structureand is never available for another binding event without denaturation ofthe amplification product.

Primer sequences appropriate for carrying out this aspect of the presentinvention are dependent upon the factors described previously for linearamplification. The segment of the primer that binds to the target mustbe of appropriate length and base composition in order to allow stablepriming at the temperature being used for the reaction. The segment ofthe primer that participates in self-hybridization after an extension ofthe primer must be of appropriate length and base composition such thata partial dissociation of the extended primer from the template issufficient for the creation of a stable secondary structure, i.e., thestem of a stem-loop structure. This structure does not have to bepermanent but only sufficiently stable such that it can allow anotherpriming event. In addition, this aspect of the invention involves thecreation of a complementary copy of the stem-loop sequences of theextended novel primer. This necessitates that the segment of the primerthat participates in self-hybridization after an extension of the primermust be of appropriate length and base composition such that thesequences involved in secondary structure can still be used astemplates. In addition to base composition and length, stability ofprimary and secondary structures can be influenced by the incorporationof modified bases into the primers, the extended sequences or both.These can either raise or lower the Tm of the segments where they arepresent. An example of a modification of a base that can raise the Tm ofa segment can be but is not limited to the addition of an EthidiumBromide moiety as described in EP O 231 495 B1. An example of amodification of a base that can decrease the Tm of a segment can be butis not limited to the use of Inosine as described by Auer et al., (1996,Nucl. Acids Res. 24; 5021-5025, cited supra, contents alreadyincorporated herein).

A non-linear amplification product can also be synthesized underisostatic or limited cycle conditions by a novel nucleic acid constructthat comprises two first segments and one second segment. Each of thefirst segments is complementary to a strand of a nucleic acid or itscomplement and the second segment is capable of forming a secondarystructure after extension of one of the first segments. This constructwould be capable of creating a product that has a pair of complementarypotential stem-loop structures. This product could be formed by acontinuous series of the following steps. One first segment and onesecond segment of the novel construct could carry out the samecontinuous series of binding, extension, secondary structure formation,regeneration of a primer binding site, second binding, second extensionand strand separation steps that have been described previously forlinear amplification by a single novel primer. In addition, the productof this synthesis could be used as a template for a series of bindingand extension steps by the other first segment as had been describedabove for non-linear amplification with a novel primer and a standardprimer. A potential series of different forms that these steps couldgenerate is given in FIGS. 5 and 6. The series of events that this novelconstruct can potentially carry out is the same as described previouslyand the final product shown in FIG. 6 is the topological equivalent ofthe final product of FIG. 2 with the two 5′ ends of the primers boundtogether.

A non-linear amplification product can be synthesized by the use of twonovel primers that are complementary to different strands of a targetnucleic acid by a continuous series of the following steps underisostatic or limited cycle conditions. Novel primer (A) binds to atarget strand and there is the same series of extension, secondarystructure formation, regeneration of a primer binding site, secondbinding, second extension and separation of the extended primer from thetemplate as described previously for linear amplification with a singlenovel primer. As extended novel primers are displaced by binding andextension of other novel primers, these single-stranded products canbind novel primer (B) and allow it to be extended to create a fulldouble-stranded amplicon. This potential series of events is depicted inFIG. 3. As described previously, the formation of the complement of anextended displaced primer creates a template with secondary structurethat should allow multiple binding, extension and displacement eventsunder isostatic or limited cycle conditions. A product can be formedthat has secondary structure at one end derived from sequencescontributed from the first novel primer and its complement and secondarystructure at the other end derived from sequences contributed by thesecond novel primer and its complement. Since this structure has a loopstructure on each strand that regenerates a single-stranded segmentcapable of being used as a primer binding site, further binding andextension of novel primers or nucleic acid constructs can be initiatedon either strand under isostatic or limited cycle conditions. Althoughfor purposes of illustration the series of events shown in FIG. 5 are aresult of a primary initiation event at one end by novel primer (A), itis understood that with the availability of the complementary templatestrand, the series of events could have been depicted in a similarfashion with a primary initiation at the primer binding site of thecomplementary target strand by novel primer (B).

Novel primers can also be modified such that the second segment isunable to be used as template while still capable of participating insecondary structure formation through self-hybridization. Means that canbe used to introduce such modifications can include but are not limitedto the inclusion of abasic sites and peptide nucleic acids. Methods ofsynthesis of such primers have been described in U.S. patent applicationSer. No. 08/749,266, cited supra and already incorporated herein. Aproduct that could be created by template dependent bindings andextensions of such novel primers or primer constructs is adouble-stranded amplicon that is capable of having in each strand asingle stem-loop at one end and a single-stranded primer binding site atthe other end.

This product can be synthesized by these modified novel primers in acontinuous series of the following steps. The first series of potentialprimer binding, extension, secondary structure formation, regenerationof a primer binding site, second binding, second extension andseparation of the extended primer from the template can be as describedpreviously for linear amplification with a single novel primer. Theseries of reactions with the second modified novel primer are shown inFIG. 6. Since it can not be used as a template, the second segment ofthe modified novel primers has no complementary strand that wouldotherwise compete against the self-hybridization of the second segmentwith the sequences created by extension, thereby allowing more efficientformation of a secondary structure. Thus, even though there is nostem-loop structure at the 3′ ends of the molecule, segments aresufficiently exposed that can be used for more additional primingevents.

A non-linear amplification product can be formed under isostatic orlimited cycle conditions by a novel nucleic acid construct thatcomprises two first segments and two second segments. Each of the firstsegments is substantially complementary to one strand or its complementand each of the second segments is capable of forming a secondarystructure after extension of one of the first segments. This constructwould be capable of forming a product that has two pairs ofcomplementary potential stem and loop structures. This product would besynthesized by one first segment and one second segment carrying out thesame continuous series of binding, extension, secondary structureformation, regeneration of a primer binding site, second binding andsecond extension steps that have been described previously fornon-linear amplification by a single novel primer. The product of thisset of reactions could then be used by the other first segment andsecond segment of the novel construct to carry out the series ofreactions described above. A potential series of different forms thatthese steps could generate is given in FIGS. 7 and 8. The series ofevents that this novel construct can potentially carry out is the sameas described previously and the final product shown in FIG. 8 is thetopological equivalent of the final product of FIG. 6 with the two 5′ends of the primers bound together. Although novel constructs with morethan one polarity have been used to illustrate various arrangements thatcan carry out linear and non-linear amplification under isothermal orlimited cycle conditions it is understood that constructs with branchedDNA can also be used for similar processes.

The compositions and methods of use of the aspects of the presentinvention that have been described above are capable of carrying outlinear or non-linear amplification without any of the limitations ofpreviously described art. In these aspects of the present invention,there is no necessity for the full cycle conditions, RNA intermediates,modified nucleotides or multiple enzymes that have been required inprevious art.

Self-propagating Novel Primers and Nucleic Acid Constructs

In all other amplification systems that have been described in previousart, no one has disclosed non-linear amplification without therequirement for two binding sites, one on each target strand. Thisrequirement is due to the necessity for the presentation of sequencesfrom each strand. Systems with this requirement have included thermalsystems such as PCR and LCR and isothermal systems such as 3SR and SDA.As such, PCR reactions are performed with two primers, Where each strandof a target nucleic acid is used by one or the other primer. Even in thedisclosure of Rose et al., two identical binding sites are required tocarry out PCR so the same primer could be used for each strand.

One aspect of the present invention discloses compositions and methodsof use for non-linear amplification where one or more binding sites fornovel primers and nucleic acid constructs are confined to only onestrand of a target nucleic acid. The novel primers and novel constructsof this aspect of the present invention have at least three segments.These segments can be joined together either covalently ornon-covalently. Means of joining segments through covalent linkages caninclude but are not limited to the phosphate backbone of normal linearnucleic acids, constructs that have more than one polarity and branchedDNA constructs. Methods of synthesis of such constructs have beendescribed in (INV patent). Means of joining segments by non-covalentlinkages can include but are not limited to ligand-receptor bonds andcomplementary base pairing. The segments may be adjacent to each otheror they may be spatially separate from each other. The sequences of thesegments may be distinct from each other or they may be complementary oridentical to each other.

When a single novel primer has a single polarity, it has three segmentswith the following characteristics:

1) The first segment of the novel primer is capable of binding andextension and comprises sequences that are substantially complementaryto sequences in only one strand of a target of interest such that it canbind to the target and be extended using the target sequence as atemplate.

2) A second segment of the novel primer comprises sequences that aresubstantially identical to sequences in the target of interest such thatthe second segment is capable of self-hybridization with sequencescreated by target dependent extension of the first segment allowing asecondary structure to form which promotes self-priming events.

3) A third segment of the novel primer is capable of acting as anintrastrand template and thereby allows self-extension.

By virtue of these characteristics, the presence of one strand of anappropriate target molecule can convert a single novel primer into aself-propagating nucleic acid capable of non-linear amplification. Thesingle novel primer of the present invention can bind to a target andutilize it as a template for extension. Due to the presence of thesecond and third segments, this product is then capable of undergoing aseries of intrastrand and interstrand binding and extension reactions.The products of these reactions are self-propagating single-strandednucleic acids or self-propagating double-stranded nucleic acids. Thesingle-stranded nucleic acid products are capable of forming stem-loopstructures and the double-stranded nucleic acids are capable of formingstem-loop structures after being rendered single-stranded.

A series of steps that can be used to synthesize such forms from linearnovel primers by the presence of the appropriate strand of a targetnucleic acid are shown in FIGS. 9, 10 and 11. A novel primer can bind toa template and be extended to form the structure of step 2 of FIG. 9where synthesis is limited to copying only a discrete portion of theavailable template. A constraint on the extent of synthesis can becarried out by a variety of means. These means can consist of but arenot limited to size, time and substrate constraints. For a sizerestraint, the target can be treated prior to extension by means thatcreate random or site-specific ends. Random sites can be used to createa pool with a select average size. Means for producing random breaks intarget nucleic acid can include but are not limited to physical methodssuch as shearing and enzymatic methods such as a nuclease. Site-specificsites can be used to create target nucleic acids that have a discretesize. Means for creating site specific ends can include but are notlimited to restriction enzymes. For a time constraint, the reaction canbe carried out for a time interval that is sufficient for the bindingand desired length of synthesis followed by an adjustment of thetemperature to stop the reaction. The duration of the time interval isdetermined by factors that can include but are not limited to buffer andsalt conditions, the choice of temperature used for binding andextension, the use of modified substrates that are used with a differentefficiency compared to normal substrates and the choice of theparticular polymerase. For substrate constraints, the primer sequencescan be chosen such that the desired extent of the extension reaction canbe carried out by a limited number of particular nucleotides andexcluding from the reaction the particular nucleotide or nucleotidesthat would allow synthesis further than the desired extent. Forinstance, omission of dTTP from a reaction mix would allow templatedependent extension of the primer with dCTP, dGTP and dATP withtermination of the growing strand occurring at the point where dTTP isrequired.

The efficiency with which extensions are stopped at the appropriatesites affects the overall efficiency of the reaction. It is understoodthat it is desirable that the stoppages be as complete as possible toinsure that the target templates have been used to produce the maximalamounts of intermediates that are capable of participating in the stepsof the reaction that will be further described for this process. On theother hand it should be noted that the constraints do not have to beabsolute in nature. As long as some of the extension reactions arelimited appropriately, reaction products are created that can undergothe further steps that are described below.

After the primer has been extended to the desired extent, the primer isseparated from its template (FIG. 9, step 3). Although not shown in thisFigure, this could potentially take place by formation of a secondarystructure through self-hybridization between the extended sequences andthe second segment of the novel primer (the a′-b′ and a-b segments ofthe novel primer shown in FIG. 9). This would allow binding andextension of other single novel primers with the same target moleculefollowed by displacement of the extended primers under isostatic orlimited cycle conditions as has been described in previous aspects ofthe present invention. However, in the absence of a design of sequencesin the novel primer that would allow this event to occur, separation ofan extended primer from its template could be carried out by thermaldenaturation in step 3 of FIG. 9, i.e., full cycle conditions. Thesequences for a-b can be chosen such that they are sequences that areadjacent to the c-d sequences in the target or as illustrated in FIG. 9they can be separated from these sequences by a segment of appropriatelength designated x-y.

After either a self-catalyzed or a thermal release step, the partiallyextended primer is capable of a self-priming event by hybridization ofcomplementary segments as shown in step 4 of FIG. 9. This allowsself-extension of the primer using the third segment of the single novelprimer as the template. When a limited subset of the four dNTP's is usedfor control of the extension length in step 2 of FIG. 9, the missingnucleotide(s) may have to be added for this further extension step.Similarly, adjustments in reaction conditions may also be needed whenfactors such as buffer, salt, temperature, polymerase or modifiednucleotides have been used to influence the time interval for thelimited extension step. The secondary extension of the primer in step 5of FIG. 9 adds sequences that are complementary to the 3′ end of anunextended primer. Step 6 shows the denaturation of the product of Step5 of FIG. 9. The extended primer can then undergo either an intra-strandself-hybridization event or an inter-strand hybridization event. Theintra-strand self-hybridization can be between the extended end andeither the first segment or the third segment of the extended primer.Self-hybridization of the extended primer with the first segment is aself-priming event that would form the structure seen in step 7 of FIG.10. This form is capable of undergoing self-extension (step 8 of FIG.10). In addition to self-propagation of sequences by these potentialintra-strand events, it also can take place by inter-strandhybridization. The binding of an initial novel primer to an extendednovel primer is shown in step 9 of FIG. 10 followed by the extension ofthe initial novel primer and the further extension of the extended novelprimer as shown in step 10 of FIG. 10. Although not shown in thisFigure, there can also be an inter-strand hybridization between extendednovel primers that could allow extension of each. Therefore, both byintramolecular and by intermolecular annealing, the extended primers canundergo the continuous addition of sequences after a denaturation event.The product of a series of reactions as depicted in FIGS. 9 and 10 wouldbe a series of amplicons with various sizes depending upon which routeof extension was taken (intramolecular or intermolecular) and how manyrounds of denaturation/extension took place.

In another aspect of the present invention, a novel primer with threesegments can be modified such that self-priming and self-extension takeplace only during the limited synthesis step and self-propagation takesplace by intermolecular bindings and extensions. This can be carried outby having a segment in the primer that partially or totally blocks itsuse as a template (FIG. 11). Methods for modifying novel primers forthis purpose have been described previously. The presence of a site thatis blocked as an extension template can still allow the same potentialseries of reactions that were shown in steps 1-6 of FIG. 9. However,after intermolecular hybridization of an initial novel primer with anextended novel primer (Step 9 of FIG. 11), only the unextended primercould have new sequences added to its 3′ end whereas the previouslyextended primer would remain the same length (step 10 of FIG. 11). Thisevent allows the further production of extended primers that can in turnbe templates for additional extension events thereby creating aself-propagating construct. In this way there can be non-linearamplification of an amplicon with a discrete size that comprises adouble-stranded segment flanked by single-stranded 5′ tails.

Constructs with Self-priming Hairpins

The formation of a self-propagating nucleic acid from a single strand oftarget nucleic acid can also be carried out by nucleic acid constructsthat comprise one or more first, second or third segments. Theseconstructs could have more than one polarity or they could be branchedDNA. In this aspect of the present invention, the segments of theconstruct have the following characteristics:

1) One or more first segments are substantially complementary tosequences in only one strand of a target of interest such that they canbind and be extended using only said strand of the target sequence as atemplate.

2) One or more second segments of the construct are substantiallyidentical to sequences in the target of interest such that they arecapable of self-hybridization with sequences created by target dependentextension of a first segment of the construct allowing a secondarystructure to form that promotes self-priming events.

3) One or more third segments of the construct are capable of acting asintrastrand templates and thereby allowing self-extension.

First segments of a construct may be substantially identical to eachother or they may be substantially dissimilar to each other. The secondand third segments may also be described in this way. Variousarrangements of sequences can be used for such constructs. For purposesof illustration, examples of such arrangements are given for constructswith multiple polarities. In this aspect of the present invention, thefinal product of template dependent bindings and extensions followed byintrastrand and interstrand bindings and extensions are constructs thatare capable of forming one or more stems and one or more loops byintramolecular hybridization.

A self-propagating nucleic acid can be formed by a novel nucleic acidconstruct that has one first, second and third segment. In this example,the second segment has its own 3′ end because it is part of a constructwith more than one polarity. However, it still functions only as asecond segment due to a blockage of the 3′ end. This blockage ofextension can be carried out by any of a number of means known to thoseskilled in the art.

A potential series of events that can take place when this construct iscontacted with an appropriate target strand is shown in FIG. 12. Afterbinding to the appropriate target strand, the first segment can undergolimited extension. The same potential means of limiting the extent ofsynthesis by size, time and substrate constraints previously describedfor limiting synthesis also find utility in processes with thisconstruct. The extended strand is then capable of either intra-strandbinding with the second segment of the same construct (step 4 a) orinter-strand binding with the second segment of another constructmolecule (step 4 b). With either of these arrangements further extensioncan take place by using the third segment as a template (step 5 a and 5b). The product of either of these processes is an extended constructthat is capable of self-propagation by being used as a template forbinding and extension of more initial primer constructs (step 6 of FIG.12).

A self-propagating nucleic acid can also be formed by a novel nucleicacid construct that has two or more first, second and third segments.Depending upon the design of the construct, a self-hybridization eventcan occur within the same extended strand or it can occur betweendifferent extended strands of the construct. Although constructs withmultiple polarity or branched DNA can physically comprise a singlestrand, for the purposes of clarity a strand in a construct refers to acontinuous stretch of nucleic acid that has a single polarity. Anexample of constructs that have two first, second and third segmentswith the strand arrangements described above are given in FIG. 13 andFIG. 15. The constructs used in this aspect of the present invention arerelated to the previous aspects that were exemplified in FIGS. 9, 10 and11. In common with these, synthesis is limited to copying only adiscrete portion of the available template. The same size, time andsubstrate constraints previously described for limiting synthesis alsofind utility in these aspects of the present invention. Thereby, step 3of FIGS. 13 and 15 are equivalent to step 2 of FIG. 9 with the exceptionthat a single template molecule is used for extension of two 3′ endsrather than only one. Release from the template can allowself-hybridization in the construct followed by further strandextension. The arrangement in FIG. 13 allows intrastrand binding andextension within a construct whereas in FIG. 15 there is inter-strandbinding and extension within a construct. For both of these arrangementsthere can be a denaturation event that can allow further self-primingand self-extension reactions by use of different copies of the repeatedsegments of the constructs. However, FIGS. 14 and 16 illustrate a seriesof events that demonstrate the ability to self-propagate by binding tounextended constructs and initiating mutual extension events.

The products of the target dependent extension reactions illustrated byFIGS. 9 through 16 are different depending upon the particulararrangement of the segments in the initial construct. However, they doshare a common secondary structure characteristic. The products of thepresent invention have intramolecular forms that constitutesingle-stranded regions as well as self-complementary double strandedregions. These single-stranded regions can be drawn as loops (theproducts of FIGS. 9, 10, 11, 13 and 14). They can be part of a circle(the product of FIG. 12). They can be part of double single-strandedloops consisting of non-complementary sequences located between twodouble-stranded regions (FIGS. 14 and 15). This characteristic is incontrast to the products of previously described constructs withmultiple polarities (INV patent) where the intramolecular products werecompletely made up of self-complementary sequences.

Although novel acid constructs have been described that used more thanone polarity for the preceding reactions it is understood that branchedDNA constructs could have been used for the same series of reactions andequivalent products. Also, the constructs with more than one polaritythat have been described in this aspect of the present invention aredifferent from those described in (INV patent). In that previouslydescribed art, the segments capable of extension were complementary toboth strands of a target nucleic acid, whereas in the present inventionthe entire reaction is carried out by template-dependent priming andextension of segments that are complementary to one and only one strandof a target nucleic acid.

Simplification of Reactions and Reaction Products

In all amplifications including the present invention, there are sidereactions that can also take place during the reaction. These can form acomplex variety of higher molecular weight products. They can bedeleterious in terms of reducing the efficiency of either synthesis ofthe desirable sequences or the efficiency of detection of thesesequences. Reduction of efficiency of synthesis can take place when theside reactions reduce the amount of synthesis of desirable sequences bycompetition for polymerase and substrates resources. The side reactionscan also reduce efficiency when appropriate sequences are synthesizedbut they are in secondary structures that are inhibitory to some of thesteps of the reactions. This can take place by structures that interferewith either binding or extension of primers. In the latter case therecan be a loss of efficiency due to an inability to use a primed templateand also due to a loss of polymerase activity if the enzyme is bound butunable to proceed. Inappropriate secondary structures can also createproblems in the detection of appropriate sequences.

Novel methods are disclosed that can be used to reduce the effects ofthese secondary reactions. These methods can be used with variousaspects of the present invention that have been previously disclosed andit is understood that they may also be used in conjunction with methodsof amplification that have been described by others. Since aself-propagating system uses products as templates for further reactionsthe extent of synthesis of any product can be controlled by reduction inthe average size by including a limited amount of terminator nucleotidesinto the reaction. In this way a product can be synthesized that cannotundergo side reaction extensions but can still be used as a template forextensions by other primers or primer constructs. This can increase theamount of appropriate sequences synthesized and reduce the amount ofpotential inhibitory elements. Abrogation of the effects of secondarystructures can also be carried out by post-synthesis methods that eithereliminate secondary structures or release the target sequences fromassociation with such structures. An example of the former method can betreatment with a single-strand specific nuclease that digests the loopsand junctions of secondary structures. Disassociation can be carried outby digestion with restriction enzymes that can isolate the desirablesegments away from other DNA sequences. Elimination and dissociation canbe carried out simultaneously by limited digestion with DNase orphysical treatments such as depurination. The product of thesetreatments would then be rendered more efficient in terms of signalgeneration by a variety of detection means.

Polynucleotides Containing Negatively Charged Modified Nucleotides

In another aspect of the present invention, methods and compositions aredisclosed that use nucleotide analogues that allow amplification ofdouble-stranded DNA targets when using denaturation temperatures thatare below those of the corresponding unsubstituted double-strandedsegment. This is carried out by the introduction of bases modified bynegatively charged constituents that reduce the Tm of an extendedproduct. In contrast to the teachings of Auer et al. describedpreviously, the substitution of the modified bases of the presentinvention base still allows temperatures for binding that are in therange commonly used with unmodified bases. The lower temperatures thatwere described by Auer et al. have the limitation that it is well knownin the art that the use of lower temperatures for binding of primers tonucleic acid targets can contribute to non-specific priming withnon-target nucleic acid templates and also to increased primer-dimerformation. The present invention avoids these limitations by retainingthe ability to use higher binding temperatures in the presence ofmodified bases. Whereas in previously described art, the differencebetween the highest and lowest temperatures used in full cycle PCR canrange between 25-50° C., the present invention can use a compressedseries of cycles that differ by less than 10° C. Thus the presentinvention provides the use of a temperature that is high enough topreserve efficient specific annealing of primers while at the same timeis low enough to avoid exposure of the enzyme and nucleotide substratesto temperatures that allow considerable levels of inactivation duringthe time used for the reaction.

Post-synthesis Labeling

In the present invention, novel compositions and methods are disclosedfor the generating non-radioactive signals that overcome the limitationsin prior art that are intrinsic to the use of large bulky groups thathave previously been used in obtaining sequence information. It has beenknown previously in the art that in general, chain terminators have aproblem being incorporated by polymerases. It has also been long knownthat the presence of large bulky groups useful in signal generationcreates a further reduction in incorporation efficiency. An example ofthis is a group of fluorescently labeled dideoxy nucleotides thatweren't substrates for the Klenow fragment of polymerase I although theycould be used by AMV Reverse Transcriptase and T7 polymerase (Prober etal., 1987, Science 238; 336-341, contents incorporated herein). Both ofthese factors can reduce the overall level of incorporation, which inturn reduces the amount of terminations and signal production. Inparticular, longer strands of DNA are adversely affected sincetermination of these loci is usually generated by reducing the amount oftermination nucleotides compared to normal nucleotides; thereby addingfurther stress on the likelihood of their incorporation.

In the present invention, these limitations are overcome by covalentlinkage of a signal generation moiety to a reactive group in aterminator nucleotide after the strand extension and termination eventsare concluded. This is in contrast to previous methods that incorporatea label either prior to or during strand elongation.

In this aspect of the present invention, reactive groups include thosethat a) provide substantially specific covalent linkage of signalingmoieties to terminal nucleotides rather than internal nucleotides and b)do not substantially inhibit incorporation of the modified terminatornucleotides or interfere with analysis by electrophoresis. Examples ofreactive groups that can be added to a terminator nucleotide can includebut are not limited to thiol, alkyl halide, free or protected primaryand secondary amine groups. Methods for creating derivatives withreactive groups can be but are not limited to those described by Ward etal., in U.S. Pat. Nos. 5,476,928; 5,241,060; 5,260,433 and 4,707,440,cited supra and already incorporated herein by reference. Groups usefulin signal generation can then be attached to the terminated strandswithout regard to any inhibitory effects upon enzymatic activity orsubstrate utilization. Groups useful in detection can include but arenot limited to haptens, ligands, receptors, fluoroscein, rhodamine,coumarin and other fluorescent molecules, infra-red fluoroscent groups,chemiluminescent moieties, energy transfer systems and enzymes. Otheruseful reactive groups include bulky or charged groups that whenincorporated into terminating nucleotides render them unusable as enzymesubstrates. Such groups include Texas Red and donor conjugates fordelayed fluorescense. Methods for attachment of signal generating groupsto reactive groups are described by Ward et al. in U.S. Pat. No.5,476,928, and also U.S. Pat. Nos. 5,241,060; 5,260,433 and 4,707,440,already incorporated herein. This aspect of the invention can be carriedout in conjunction with methods disclosed previously for production ofmultiple copies from a single template under isostatic or limited cycleconditions. In addition, it is understood that post-polymerisationlabeling can also be carried out when using any means that have beendescribed previous to the disclosure of the present invention.

This method is in contrast to the original description of using thechain terminator as a source of signal generation as described by Hobbsand Cocuzza in U.S. Pat. No. 5,047,519 (incorporated herein) teach awayfrom the present invention where they explicitly state that “To beuseful as a chain-terminating substrate for fluorescence-based DNAsequencing, a substrate must contain a fluorescent label . . . ”. Thepresent method overcomes the limitation that either dNTPs or ddNTPs haveto be marked prior to incorporation in any of the commonly employedmeans of sequence analysis of labeled DNA strands. These can includeboth static systems and real time analysis. Examples of static systemswould include but not be limited to acrylamide gel separations followedby photography or chemiluminescence detection. Examples of real timeanalysis would include but not be limited to acrylamide gel separationsfollowed by detection of a single dye as used by Ansorge et al., 1986 orit could be distinct dyes for each base termination as described bySmith et al., in U.S. Pat. No. 5,171,534 and Prober et al., in U.S. Pat.No. 5,332,666. The contents of the foregoing publication and two patentdocuments are incorporated herein by reference. It is also understoodthat although the present invention has been described in terms of chaintermination by dideoxynucleotides, other chain terminators can also beused. A description of various chain terminators is given in “DNAReplication”, 2^(nd) Edition, by A. Kornberg and T. A. Baker, 1992,447-449, W. H. Freeman and Co., NY, N.Y., incorporated herein. Examplesof changes in the sugar ring can include but not limited to acyclo andarabinosyl dNTPs. When used as terminal nucleotides these derivativesmay be of particular use since chemically and biochemically they shouldbe well distinguished from the normal nucleotides that comprise theother parts of the DNA strands. It has also been shown in U.S. Pat. No.5,332, 666 (incorporated herein) that fluorescently labeled acycloderivatives can produce sequence ladders that are equivalent to onesderived by radioactive labeling. In addition, blockage of chaintermination by the presence of an amino group in the 3′ position of aterminator nucleotide can also provide a functional group for thepost-synthesis attachment of a signal generating moiety. Other blockinggroups can be used that are capable of regenerating an active 3′-OH endof a strand. For instance when a photocleavable group is included asdescribed in the art by the 3′ OH can be regenerated after the reactionhas been terminated and used for attachment of a label. Systems thatcould be used for this function include but are not limited toincorporation of a fluorescently labeled dideoxynucleotide by terminaltransferase.

Another aspect of the present invention is directed towards overcominglimitations inherent in the primer labeling system by separation of theprimer labeled extension products that have been terminated properlyfrom those which have not. Such a separation can be achieved by usingproperties of modified terminator nucleotides. This can be carried outby either a pre-existing marker in the terminator nucleotide or by apost-synthesis modification as described above. Any means that can allowa suitable physical separation between the presence and absence of amarker is considered to be within the scope of the present invention.Examples of such pre-existing markers can consist of but not be limitedto biotin, imino-biotin, fluorescein, halogens, thiols, and amines.Means of physically sequestering strands that have such markers canconsist of but not be limited to avidin, streptavidin, antibodies andphysical matrices that combine with halogens, thiols or amines. Afterseparation of the marked strands from the strands lacking terminatornucleotides, the products can be released in a form that is suitable forsequence. Examples of means for such release can consist of but not belimited to physical denaturation of proteins such as antibodies throughheat or chemical treatments. Release can also be carried out by use of ascissable bond such as a disulfide bridge or imino biotin. Methods ofuse of scissable bonds is described in the Ward disclosures cited supra.Signal generation from the purified strands can be carried out bymarkers in the oligonucleotide used as a primer, or in the dNTP or ddNTPnucleotides that have been added to the primer.

The processes of the present invention can be adapted to signalgeneration for all sequencing procedures including those that use asingle channel and 4 different dyes as well as procedures that use 4channels and a single dye. Signals produced in such procedures can beanalyzed in real time of by scanning.

Thus, the present invention provides a post-termination labeling processfor nucleic acid sequencing comprising three steps. First, nucleic acidfragments corresponding to the nucleic acid sequence of interest areproduced in the presence of untagged or unlabeled substrates, untaggedor unlabeled primer, polymerizing enzyme, buffer and an appropriateuntagged or unlabeled terminator for each nucleotide base. Each of theterminators comprise a chemically reactive group that covalently bindsto a tagged molecule under conditions that internal sequences aresubstantially non-reactive to the tagged molecules and the chemicalreactions do not substantially interfere with separation of thefragments in a medium or matrix. Next, the fragments produced in amedium or matrix are separated followed by detection of the separatedfragments by means of detecting the tagged molecule in said medium ormatrix.

Various embodiments may be included in the above-describedpost-termination labeling process. In the producing step, for example,the chemically reactive groups of the terminators can be protected priorto their enzymatic incorporation into the fragment produced and they canthen be deprotected prior to covalently binding any tagged molecule. Thechemically reactive group can comprise a nitrogen, a sulfur or an oxygenatom. Furthermore, the chemically reactive groups on said terminatorscan be different, or they can be the same. In addition, the taggedmolecule can be the same or they can be different for each terminator.Those skilled in the art will readily appreciate that such taggedmolecules are known and may be selected from the group consisting offluorescent dyes, chemiluminescent dyes, infra-red dyes,chemiluminescent entities and electrochemiluminescent entities, orcombinations thereof.

In addition to the just described embodiments and features of thepost-termination labeling process, the separating step can be carriedout electrophoretically, and the medium or matrix can comprise a gel,such as a polyacrylamide gel. Separation can also be carried out bycapillary gel electrophoresis.

With respect to detection, this step can be carried out by a meansselected from photometric measurement, spectrophotometric measurement,calorimetric measurement, fluorometric measurement, delayed fluorescentmeasurement and chemiluminescent measurement, or combinations thereof.

Utility of Invention

The various aspects of the present invention that are capable ofcarrying out linear and non-linear amplification of nucleic acidsequences can fulfill many of the functions that have previously beencarried out by methods described in previous art for isothermal andthermocycler dependent methods. These can include but are not limited tosequencing, probe synthesis and labeling, forensic identification,allele identification, genetic screening, isolation and cloning ofdesirable genes, artificial gene construction, gene expression anddiagnostic identification. Reverse Transcriptase or a DNA polymerasecapable of reverse transcription can be used to practice the presentinvention with an RNA molecule as the initial substrate. The reactionscan be carried out in the absence of any modifications of the primers orreagents or if desired these can be labeled or otherwise modified. Thepresence of amplified sequences can be assayed directly either byincorporation of labeled moieties or by direct visualization. Indirectmeans of identification can also be carried out by hybridization withappropriate probes. These indirect means can include but are not limitedto dot blot, slot blot, Southern blot and plate assay formats.

Although certain nucleic acid sequences are required to be present inthe primers for binding to template or to create self-complementaryregions for multiple priming events, additional nucleic acid sequencescan be included in the primer sequences to provide desirable propertiesby their presence. These can include but are not limited to phage RNApromoters to allow a further amplification of desirable nucleic acidsequences and sequences that can be used for identification or isolationof amplicons. Sequences could be included in primers or a primerconstruct that would create an amplicon with an inverted repeat at eachend. This segment could then be used as a binding site for a singleprimer or primer construct that could use either strand for binding andextension. Since the choice of the inserted sequence is arbitrary, thissegment could be a universal target that could be used for amplificationregardless of the sequences in between.

Also provided by the present invention are processes for producingnucleic acid sequences that have decreased thermodynamic stability tocomplementary sequences. In this process, at least one modifiednucleotide or nucleotide analog having a negatively charged chemicalmoiety is incorporated into the nucleic acid sequences produced.

A further provision of the present invention is a single-stranded ordouble-stranded nucleic acid polymer selected from the group consistingof a linear nucleic acid, branched nucleic acid, an inverted nucleicacid and a peptide-nucleic acid, or a combination of any of theforegoing. The nucleic acid polymer comprises at least one purine orpyrimidine base comprising one negatively charged chemical moiety in oneor both strands of the polymer.

This invention further contemplates and embraces compositions and kitsfor use in the variously described processes above.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1

Isothermal Amplification of PCR Product by Bst Polymerase at 53° C. and63° C.

(i) PCR Amplification of HBV Plasmid DNA

The HBV positive control from the HBV Microtitre Plate Assay (ENZODiagnostics, NY, N.Y.) was used as a target for amplification by PCR.According to the manufacturer, this DNA is 80 pg/ul (the equivalent of1.2×10⁷ copies of HBV per ul). A 50 ul PCR reaction was carried outconsisting of 1 ul of HBV target, 1×PE buffer (Perkin-Elmer, Emeryville,Calif.), 4 mM MgCl₂, 250 um dNTP, 6 units of Amplitherm (Invitrogen,LaJolla, Calif.) and 10 pMoles of HBV oligo primers FC and RC.

FC Sequence=5′-CATAGCAGCA GGATGAAGAG GAATATGATA GGATGTGTCTGCGGCGTTT-3′  (Sequence 1)

RC Sequence=5′-TCCTCTAATT CCAGGATCAA CAACAACCAG AGGTTTTGCATGGTCCCGTA-3′  (Sequence 2)

In this example, the 29 bases at the 3′ end of the FC primer and the 30bases at the 3′ end of the RC primer are first segments that are capableof extension using HBV target DNA as a template. The 30 bases at the 5′end of the FC and RC primers are second segments that are complementaryto the first 30 bases synthesized by extension of the primers using HBVDNA as a template. Thermocycling conditions were 30 cycles of 94° C. for1′, 56° C. for 15″, and 68° C. for 30″. Based on the HBV sequence, theanticipated PCR product should be 211 bp in length. Stem-loop structuresare possible at each end of this product with 30 base pair stemscontributed by the second segment and its complement and 29 and 30 baseloops contributed by the FC and RC first segments respectively.

(ii) Analysis of PCR Product

The amplification was assayed by gel electrophoresis of a 10 ul samplein a 4% Metaphor agarose gel (FMC BioProducts, Rockland, Me.) that wasrun with 0.5×TBE buffer in the presence of 0.5 ug/ml Ethidium Bromide.Under UV illumination, three bands appeared that as judged by DNA sizemarkers were approximately 210, 180 and 170 bp in length. The bandcorresponding to 210 bp is the linear PCR product that had beenanticipated and presumably the other two bands correspond to the samesize amplicons where secondary structures are formed on either one orboth ends thereby changing their effective mobilities.

iii) Isothermal Amplification of the PCR Product

5 ul of various dilutions of the PCR product from above were used in a100 ul reaction mix consisting of 1×ThermoPol buffer (NE Biolabs,Beverly, Mass.), 200 uM dNTP, 20 pMoles of Forward and Reverse Primers,8 units Bst Polymerase (NE Biolabs, Beverly Mass.). The Forward Primerswere either FC or LFC, the Reverse Primers were either RC or LRC. Thesequences of the FC and RC primers have been given above. The LFC andLRC primers have sequences that correspond to the first segments only ofthe FC and RC primers. As such their sequences are as follows:

LFC=5′-GGATGTGTCT GCGGCGTTT-3  (Sequence 3)

LRC=5′-AGGTTTTGCA TGGTCCCGTA-3′  (Sequence 4)

Incubations were for 30 minutes, 180 minutes or overnight incubations.Temperatures for the reactions were either 53° C. or 63° C. The 30minute reactions were analyzed by gel electrophoresis with a 2% agarosegel; the 180 minute reactions were analyzed with 4% Metaphor agarose.

The results of this analysis are shown in FIG. 17. In the first set ofsamples taken after 30 minutes of incubation, only the 10⁻² dilution ofthe PCR product shows any synthesis at 53° C. whereas the reactions fromthe 63° C. show synthesis in the 10⁻², 10⁻³ and 10⁻⁴ dilutions. Thesedata demonstrates that the amount of synthesis is dependent upon theamount of input target DNA. In the set of samples taken after 180minutes of synthesis there is substantially more synthesis. The productof these reactions is a series of bands that form a discrete pattern.This is in contrast to a single discrete band that is usually seen inPCR or the two or three bands seen previously with the LC and RC primersafter PCR amplification. This multiplicity of bands may possibly be dueto the presence of the secondary structures allowing the amplicons tofunction as primers as well as templates or it may be an indication ofstrand switching. After incubation at 53° C. for three hours, even thecontrol without any target shows evidence of substantial synthesis.However, it can be noted that there is a single target dependent patternthat is seen in all the 53° C. reactions with target template and thepattern present in the no target control is substantially different,presumably due to having a different pathway from the target initiatedsynthesis. The 63° C. incubations show substantial synthesis at alldilutions of the template and demonstrate the same pattern produced bythe 53° C. reactions. However, in this experiment, there was no evidenceat 63° C. for target independent amplification. The presence ofsubstantial amounts of synthesis at even the 10−⁵ dilution is anindication that the system is capable of substantial amplification. Theovernight incubations were also analyzed by gels and showed the samepatterns and amounts as the 3 hour incubations (data not shown).

Example 2

Timecourse/sensitivity of Isothermal Amplification of HBV Sequences

i) Amplification

HBV plasmid DNA previously digested with Eco R1 was used as a templatefor isothermal amplification. DNA Mixtures consisted of 100 ulcontaining 40 pM each of FC and RC primers, 1×ThermoPol Buffer (N.E.Biolabs, Beverly Mass.) and 4×10⁶, 4×10⁴ or 0 HBV molecules. These wereheated to 94° C. for 5 minutes using a Model 480 thermocycler fromPerkin-Elmer (Emeryville, Calif.). The machine was then set for 480minutes at 63° C. After the block had adjusted to 63° C., individualtubes containing 25 ul of Enzyme Mix were put into the thermocyclerblock. Each tube of Enzyme Mix contained 4 units of Bst polymerase (N.E.Biolabs, Beverly Mass.), 1×ThermoPol Buffer (N.E. Biolabs, BeverlyMass.) and 400 uM dNTP's. After the DNA Mixtures and Enzyme Mixes hadadjusted to 63° C., 25 ul samples were taken from each. DNA Mixture andadded to each Enzyme Mix tube for a total volume of 50 ul each. For eachtube of DNA Mixture, three samples were taken. One sample for each DNAconcentration was taken out of the 63° C. block after 2, 4 and 8 hours.

ii) Assay for Amplification

To distinguish between target dependent amplification and targetindependent amplification, microtitre plate assays were used to detectthe presence of target specific sequences. The reagents and directionsof use for this assay were taken from the HBV Microtitre Plate Assayfrom ENZO Diagnostics (Farmingdale, N.Y.) with the substitution ofplates and signal probes specific for the amplicon made by LC and RCprimers.

iii) Preparation of Microtitre Plates

Plates were prepared in a batch process that used 5 frames with 12(Dynel?) strips (manufacturer) in each. The sequence of the captureoligonucleotide used for these plates was derived from a region of HBVthat is in between the FC and RC primers and is described as follows:

5′-CTCATCTTCT TATTGGTTCT TCTGGATTAT CAAGGTAT-3′  (Sequence 5)

Each well of the microtitre plate was rinsed twice with 200 ul of 1 MAmmonium acetate and then left inverted at room temperature for 2 hours.A 10 ul solution containing 100 uM of the capture oligonucleotidedescribed above was mixed with 27.5 ml of 1 M Ammonium acetate. 50 ul ofthis solution was added to each well and the plates were incubatedovernight at 37° C. in an incubator with an open container of 1 MAmmonium acetate. The next day, each well was washed once with 200 ul of1 M Ammonium acetate and the plates dried overnight. Strips that werethen placed in a pouch with dessicant for future use.

iv) Preparation of Probe

The RC oligonucleotide used as a primer for the amplification was alsoused as the signal probe for the plate assay. T-tailing of 100 uM of theRC oligonucleotide was carried out by use of a Terminal Tailing kit fromENZO Diagnostics (Farmingdale, N.Y.). 26 ul of the tailed RColigonucleotide was mixed with 12.8 ml Signal probe buffer (33%Deionized Formamide, 5 mM EDTA (pH 8.0), 1% Triton X-100, 2.5% DextranSulfate, 0.15M NaCl, 0.12M HEPES (free acid), 0.01% phenol Red).

e) The results of the microtitre plate assay with the samples from thereactions are given below:

2 Hours 4 Hours 8 Hours 1 × 10⁶ targets 0.413 1.491 1.419 1 × 10⁴targets 0.203 0.098 1.017 No target 0.086 0.085 0.063

As can be seen above, the amount of product was related to both theamount of initial target and the amount of time the reaction proceeds.There was also no signal generated from any products formed in theabsence of target. Also, in this assay values of 1.4 or greater aresaturation values and the amount of product can be much greater.Assessment of the total amount of product would require dilutions of theproduct until it was in the dynamic range of the assay. However, for thepurposes of this example this was not done. The amplification reactionspreviously described are not dependent upon the use of Bst polymerase.When a different enyzme, Bca polymerase (PanVera, Madison, Wis.) wassubstituted, substantial amounts of synthesis could also be seen in theplate assay (data not shown). In addition, the temperature maximum forthe Bst reaction appeared to be 63° C. but when the Bca polymerase wassubstituted, amplification could be achieved at 68° C. According to theliterature that accompanied each enzyme, the optimal temperature for theBst and Bca polymerases is 65° C. and 70° C. respectively. This mayaccount for the 5° C. differences in their maximal temperatures.

Example 3

Amplification with ΔTth Polymerase

Due to the heat lability of the the Bst or Bca polymerases, thereactions in the previous examples, had to be carried out in two steps.The denaturation of target DNA was carried out in the absence of thepolymerase followed by addition of the enzyme after equilibration at alower temperature. It would be desirable to be able to include thepolymerase in the initial step so as to reduce the handling steps and toreduce the chances of amplicon carryover contamination. Therefore,conditions were established that allowed the use of a 5′-3′ Exo⁻derivative of Tth polymerase to carry out isothermal amplfication. Inthis particular example, two primers designated FJ and RJ were used thathad the following sequences:

FJ Sequence=5′-CATAGCAGCA GGATGAAGAG GAATATGATA GCT GGATGTGTCTGCGGCGTTT-3′  (Sequence 6)

RJ Sequence=5′-TCCTCTAATT CCAGGATCAA CAACAACCAG TGC AGGTTTTGCATGGTCCCGTA-3  (Sequence 7)

Each of these primers are similar to the FC and FJ primers describedpreviously except that they each have three more nucleotides (underlinedabove) in their first segments. Reactions were set up with 1×10⁶, 1×10⁴,or 1×10² HBV target molecules. One reaction contained 50 ng of T7 DNAinstead of HBV DNA and this was used as the no target control. Reactionconditions were as follows: limit the Isothermal amplification had to becarried out in two phases the first step was a high temperature 94° C.denaturation of target molecules.

Amplification with ΔTth Polymerase

Due to the heat lability of the the Bst or Bca polymerases, thereactions in the previous examples, had to be carried out in two steps.The denaturation of target DNA was carried out in the absence of thepolymerase followed by addition of the enzyme after equilibration at alower temperature. It would be desirable to be able to include thepolymerase in the initial step so as to reduce the handling steps and toreduce the chances of amplicon carryover contamination. Therefore,conditions were established that allowed the use of a 5′-3′ Exo⁻derivative of Tth polymerase to carry out isothermal amplfication. Inthis particular example, two primers designated FJ and RJ were used thathad the following sequences:

FJ Sequence=5′-CATAGCAGCA GGATGAAGAG GAATATGATA GCT GGATGTGTCTGCGGCGTTT-3′  (Sequence 6)

RJ Sequence=5′-TCCTCTAATT CCAGGATCAA CAACAACCAG TGC AGGTTTTGCATGGTCCCGTA-3′  (Sequence 7)

Each of these primers are similar to the FC and FJ primers describedpreviously except that they each have three more nucleotides (underlinedabove) in their first segments. Either isothermal or PCR reactions werecarried out with 1×10⁶, 1×10⁴, or 1×10² HBV target molecules in a 50 ulreaction volume. A no target control was also included that contained 50ng of T7 DNA instead of HBV DNA. Each target concentration was setup ina 100 ul reaction mixture that contained 10 units of ΔTth polymerase(Clontech Laboratories, Palo Alto, Calif.), 1×ΔTth polymerase buffer(Clontech Laboratories, Palo Alto, Calif.), 250 uM dNTP, 2.5 mM MgCl₂,1×PCR Enhancer (Epicentre Technologies, Madison, Wis.) and 20 pmoleseach of FJ and RJ primers. Each mixture was divided into two 50 ulportions. One 50 ul portion was used in an isothermal reaction byheating to 94° C. for 5 minutes followed by 240 minutes at 68° C. in athermocycler. The other portion was maintained at 4° C. until theisothermal reaction was finished and the thermocycler was used to carryout PCR with this portion with 35 cycles of 94° C. for 1 minute and 68°C. for 45 seconds.

The extent of the isothermal reaction was measured by gelelectrophoresis and plate assays as described in the previous examples.The plate assay was carried out as described previously except that theT-tailed probe was derived from the LFC primer. Results from each ofthese methods are shown in FIG. 18. The gel electrophoresis showsextensive synthesis with the 1×10⁶ and 1×10⁴ target reactions afterisothermal amplification. Although it does not show up well in aphotograph, the gel also showed a lesser level of amplification with the1×10² target reaction. The PCR reactions were also examined in the samegel and show essentially similar results with high levels ofamplification with the 1×10⁶ and 1×10⁴ target reactions. Under theseconditions there is also seen a non-specific reaction that created asmaller amplicon that increased inversely with the amount of synthesisof the appropriate amplicon. When the isothermal reactions were alsoassayed by the plate assay, the higher levels of targets gave saturationlevels and it can be clearly seen that the 1×10⁴ target level gave apositive reaction. It should be noted that the negative control showedno signs of signal generation by either of the two assays.

Example 4

Use of a Single Primer for Amplification

Each sample consisted of a 50 ul reaction containing 1×Taq Buffer(Perkin Elmer, Emeryville, Calif.), 5 mM MgCl₂, 200 uM dNTPs, and 5units of Amplitaq Gold (Perkin Elmer, Emeryville, Calif.). Each reactionalso had 5 pM of an oligonucleotide primer with the following sequence:

5′CCTGCTGCTA TGCCTCATCT GACAAACGGG CAACATACCT CCTGCTGCTATGCCTCATCT-3′  (Sequence 8)

Single primer amplifications were carried out in duplicate with orwithout target DNA (1 ul of the control HBV described previously inExample 1). Reactions were carried out in a thermoycler with one cycleof 94° C. followed by 50 cycles of 94° C. for 1 minute, 60° C. for 15seconds and 68° C. for 15 seconds. To reduce non-specific priming,samples were not added to the thermocycler block until it reached 90° C.during the first cycle.

The extent of the reactions was analyzed by microtitre plate assaysusing the same plates, probe and format described in Example 2. Theresults of the reactions (in duplicate) were as follows:

HBV+ 1.407 0.377 No Target 0.083 0.087

As can be seen above, although there was some variation in the amount ofsignal generated from the duplicate reactions, there was a clearindication of the presence of target sequences after carrying outamplification with a single primer.

Example 5

Primer Extension with Carboxy-dUTP

i) Synthesis of Carboxy-dUTP

A 5 ml solution containing 100 uMoles of allylamine-dUTP (ENZODiagnostics, Farmingdale, N.Y.) in 0.2 M Sodium Borate buffer (pH 9.2)was mixed with a 20-fold molar excess of Succinic Anhydride (Aldrich,Milwaukee, Wis.) dissolved in 5 ml of Dichloro Methane (Aldrich,Milwaukee, Wis.). This suspension was transferred to a 50 ml Falcon tubeand vortexed. The pH of the aqueous phase was readjusted to a value ofof 9.2 by addition of appropriate amounts of Triethylamine (Aldrich,Milwaukee, Wis.). Shaking and readjustment was continued until the pHvalue stabilized. An aliquot was taken and tested by HPLC for thedisappearance of the allylamine peak and the appearance of a later peakthat represented the carboxy-modified product. The aqueous phase wasremoved and diluted 10-fold with H₂O and loaded onto a DEAE-SephadexA-50 column pre-equilibrated with 0.05M Triethylammonium Bicarbonatebuffer (pH 7.8). The product was eluted by a 0.05 M-0.70 M gradient ofTriethylammonium Bicarbonate. Fractions were collected and analyzed byUV absorption at 290 nM. Appropriate fractions were checked by HPLC forpurity. Fractions with >99.5% purity were pooled together and saltsremoved in a rotary evaporator in vacuum at 30° C. The remaining solidswere dissolved in an appropriate amount of H₂O and adjusted to a finalconcentration of 10 mM as judged by absorption at 290 nM. Aliquots wereprepared and store at −70° C. until used.

ii) Primer Extension Reactions

The template for primer extension was single-stranded DNA obtained byPEG precipitation of phage particles from an mp18 clone that contained a1.4 kb insert of HBV. The primer for extension was PM-1 whose sequenceis complementary to part of the lac region of the mp18 vector. Thesequence from this primer was:

5′-CGC CAG GGT TTT CCC AGT CAC GAC-3′  (Sequence 9)

A 300 ul DNA Mixture was made that contained 7.5 ug of thesingle-stranded DNA and 60 pM of the PM1 primer. Separate 25 ul Enzymemixes were made that contained 0.5 ul of polymerase, 2×buffer and 200 nMdNTP where for each condition one reaction had normal TTP and onereaction had carboxy-dUTP. 25 ul of the DNA Mixture was mixed with 25 ulof an Enzyme mix and incubated at the appropriate temperature for 30minutes.

The following polymerases were used in this example: Exo(−) Klenow (Qunits/ml from NE Biolabs, Beverly, Mass.), Taq polymerase (T units/ulfrom GIBCO BRL, Gaithersburg, Md.), and Bst polymerase (4 units/ul fromNE Biolabs, Beverly, Mass.)

Buffers and their compositions are as follows:

1×NEBuffer 2 (N.E. Biolabs, Beverly, Mass.) consists of 10 mM Tris-HCl,10 mM MgCl₂, 50 mM NaCl and 1 mM DTT (pH 7.9 at 25° C.)

Buffer 2A was the same as NEBuffer 2 except the pH is 7.1 and the MgCl₂was only 2 mM.

Buffer 2M was the same as NEBuffer 2 except the MgCl₂ was only 2 mM and1 mM MnSO₄ was also included.

1×ThermoPol Buffer (N.E. Biolabs, Beverly, Mass.) consists of 20 mMTris-HCl (ph 8.8 at 25° C.), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄ and0.1% Triton X-100.

iii) Analysis of Primer Extension Reactions

Assessment of the ability of the various polymerases to utilize thecarboxy-dUTP as a substrate can be carried out in a number of differentways. In the present example, this was qualitatively observed withoutthe use of labeled precursors by evaluation of the conversion of thesingle-stranded DNA into a double-stranded form by gel analysis. Thisconversion event can be seen by a retardation in its migration in anagarose gel compared to the single-stranded precursor and by anincreased fluorescence due to its ability to bind Ethidium Bromide moreefficiently. Although this method was used in the initial assessments ofstudies on carboxy-dUTP incorporation, more information can be obtainedby digesting the extension products with a restriction enzyme. Since theparticular restriction enzyme used for this analysis is unable to digestsingle-stranded DNA, production of fragments is an indication of adouble-stranded region at the restriction enzyme site. By reduction ofthe circular DNA into linear pieces, it becomes easier to makecomparative assessments of the amount of conversion by the differentpolymerases. In addition since the positions of the various restrictionfragments are known relative to the primer, it allows an evaluation ofthe length of the extended product.

iv) Digestion with Restriction Enzymes

Since the templates from the primer extension reactions consisted ofunmodified DNA, the carboxy-dUTP reactions contain one strand that isnormal and a complementary strand that is substituted completely withthe carboxy-dU derivative producing a hemi-substituted restriction sitewherever T's are part of the recognition site. The enzyme used for theevaluation was BstN1 whose recognition sequence is GG A/T CC. Thecomputer program MacDNASIS (Hitachi Software Engineering America, Ltd;South San Fransisco, Calif.) was used to predict the locations of theindividual GG(T)CC and GG(A)CC sites.

v) Analysis of Reactions

FIG. 19 shows the results of extension reactions with various buffers,enzymes and temperatures. First off it can be noted that the reactionswith the unsubstituted dNTP form a different pattern from the reactionswith carboxy-dUTP. Analysis of the positions of the GG(T)CC and GG(A)CCsequences in the products showed that the pattern from BstN1 digestionof the unsubstituted reactions was due to the expected digestion at boththe GG(T)CC and GG(A)CC sites but the carboxy-dUTP reactions onlyexhibited digestions at the GG(A)CC sequences. A reaction was alsocarried out using dUTP as a substrate and there was digestion at allsites showing that it was the presence of the carboxy and its linkerrather than the use of dU that was the cause of the resistance todigestion by BstN1 (data not shown). FIG. 20 is a compilation of theresults from FIG. 19 with relative levels of synthesis rated from (−)for no synthesis, (+/−) for barely visible and on up to a rating of(++++). In general the best synthesis for carboxy-U incorporation wasseen with the Bst polymerase/Thermopol Buffer conditions.

Example 6

Effects of Carboxy-U on Mg++ Requirements in PCR

PCR amplification was carried out with double-stranded T7 DNA as atemplate and two oligonucleotides TS-1 and TS-4 as primers. Theseoligonucleotides have been previously described in U.S. patentapplication Ser. No. 08/574,443, filed on Dec. 15, 1995 and produce a622 base pair product. 100 ul reactions were carried out that consistedof 400 ng of T7 DNA, 50 pM of TS-1, 50 pM of TS-4, 1×PE Buffer (BRL) 200mM dNTP, and 15 units of Taq Polymerase (GIBCO BRL, Gaithersburg, Md.).Cycle conditions were 25 cycles of 94° C. for 50 seconds, 50° C. for 25seconds and 68° C. for 3 minutes. FIG. 21 shows the results of thissynthesis. When normal nucleotides were used as substrates for thereaction, 1 mM MgCl₂ was adequate for amplification. In contrast, whenthe reaction was carried out with carboxy-dUTP, 2 mM MgCl₂ produced onlydimers of the oligos and a minimum of 3 mM MgCl₂ was necessary forsynthesis of the appropriately sized amplicon.

Example 7

Various Thermostable Polymerases

Amplification was carried out as described in Example X-22 except thatall reactions were carried out in the presence of 3 mM MgCl₂ and only 5units of polymerase was used for each reaction. Taq polymerase (GIBCOBRL, Gaithersburg, Md.) was compared to Tfl, Tth, Amplitherm andReplitherm polymerases (all from Epicentre, Madison Wis.). All reactionsused the buffers that came with each enyzme. Gel analysis of thereactions is shown in FIG. 22. Lowering the amount of Taq gave aconsiderable reduction of synthesis in the presence of the carboxy-UTPcompared to the reaction seen in Example 6. There was no effect at allseen with the normal TTP. For the other polymerases, the only one thatgave any appreciable amount of product was the Tth polymerase and thiswas more active than the Taq polymerase under the conditions used.

Example 8

In Example 7, the reactions with the various polymerases were carriedout in the presence of 3 mM MgCl₂. However, the lack of synthesis bysome of these polymerases may reflect a different Mg++ concentrationrequirement when carboxy-UTP is a substrate. One of the enzymes thatshowed no synthesis in the presence of carboxy-UTP but gave extensivesynthesis with normal TTP was the Tfl polymerase. This enzyme was triedunder the same reaction conditions described above but 2 mM, 4 mM and 6mM MgCl₂ levels were used for the reactions. In addition, the sametitration was used with Taq polymerase (Perkin-Elmer, Foster City,Calif.) in PE Buffer (Perkin-Elmer, Foster City, Calif.) and with Tflpolymerase with the addition of 5 ul of PCR Enhancer (Stratagene, LaJolla, Calif.). The results of this are shown in FIG. 23. Under theconditions used, 6 mM MgCl₂ gave the best amount of synthesis for theTaq polymerase and the Tfl alone showed no synthesis with 2, 4 or 6 mMMgCl₂. However, when PCR Enhancer was included in the reaction, the Tflpolymerase was able to generate appreciable amounts of synthesis.Similar to Taq, the highest level was achieved with 6 mM MgCl₂. Thelevel of synthesis shown for the Tfl/PCR Enhancer reaction was alsohigher than the Taq reaction. PCR Enhancer was tried with the otherthermostable polymerases Tth, Amplitherm and Replitherm foramplification with the carboxy-UTP. The results of this are shown inFIG. 24. Although it was unable to rescue amplification by theAmplitherm polymerase, there was now synthesis shown for the Replithermpolymerase. The Tth polymerase, which other than Taq was the onlypolymerase to show amplification with the carboxy-UTP in Example 7,showed the highest level of amplification with the PCR Enhancer. Also,for the Tth/PCR Enhancer series, the reaction with 8 mM MgCl₂ gave moreamplification than 6 mM MgCl₂ reaction.

Example 9

Variations in the Thermal Conditions of Amplification

Two oligonucleotides, TS13 and TS14, that have also been described inENZ (?) were used for amplification of a different segment ofbacteriophage T7 DNA in the presence of carboxy-dUTP. The product ofthese primers is a 136 bp amplicon that is smaller than the productsynthesized in the previous examples. A sequential series of PCRreactions were carried out in two phases. Each reaction used the Tthpolymerase as well as the PCR Enhancer described above. The first phasein each reaction was a series of 5 cycles using the cycling conditionsdescribed above to create templates containing carboxy-dU in bothstrands. The second phase in each reaction used various lowertemperatures for the annealing, elongation or denaturation steps.Preliminary attempts at varying temperatures showed that temperaturesbelow 80° C. were unsuccessful in carrying out amplification with thisamplicon so that efforts to close the difference between the highest andlowest temperature were made by raising the annealing temperature. Foreach set of temperature conditions, the MgCl₂ level was also varied. Acompilation of results derived from a gel analysis of three sets ofthese reactions is given below:

Denaturation Annealing Extension MgCl₂ Synthesis a) 80° C. 2′ 55° C. 25″68° C. 2′ 5 mM ++ 4 mM + 3 mM +/− b) 80° C. 2′ 60° C. 25″ 68° C. 2′ 6 mM− 5 mM − 4 mM ++ c) 80° C. 2′ 65° C. 25″ 70° C. 2′ 6 mM − 4 mM ++ 2 mM++

In addition to the reactions above, a series of reactions was carriedout with a denaturation step of 80° C. for 2′ combined with anannealing/extension step of 68° C. for 2′. Various factors were alsoincluded with this compressed cycle to see if the efficiency of thereaction could be augmented. A gel with the products from thesereactions is shown in FIG. 25. It can be seen that with as small adifference as 12° C. between the highest and lowest temperatures therewas still amplification of the amplicon and the only factor that seemedto enhance synthesis under these thermal conditions was the addition ofextra polymerase.

Example 9

Effect on Compressed Thermal Amplification Conditions by Variations inthe Primer Sequence

In addition to the TS13 and TS 14 primers described above, primers weredesigned that had variations of these sequences to see if the rangebetween the highest and lowest temperatures could be further compressed.The sequences for TS13, TS14 primers and their variants as well as theregion of the T7 genome from which they were derived is shown in FIG.26. The differences in the sequences of these primers made some smallchanges in the size of the amplicon but essentially the same T7 segmentwas amplified in each of the reactions with these primers.

A series of reactions were carried out using various combinations of theprimers from FIG. 26. With 20 cycles of 80° C. for 2′ and 68° C. for2′30″, there was only a 12° C. separation between the denaturation stepand the annealing/extension step. A gel analysis of these reactions isshown in FIG. 27. All of the primer combinations demonstratedamplification of an appropriate band although there were difference intheir efficiencies. Controls were also included in this set of reactionswhere either normal dTTP or allylamine-dUTP was used in place of thecarboxy-dUTP; these reactions gave no detectable levels ofamplification.

The same combinations of primers were tried in amplification reactionswith 20 cycles of 80° C. for 2′ and 72° C. for 2′30″. A gel analysis ofthese reactions is shown in FIG. 28. Under these thermal conditions,most of the primer combinations failed to amplify. However, all of thereactions that included the TS23 primer as one of the primer pairs gaveamplification. In regard to the other primers that were used inconjunction with the TS23 primer, the relative levels of amplificationwere in the order of TS21>TS22>TS13. Although there may be other factorsinvolved, this ordering may be related to an inverse relationship to thenumbers of carboxy-dUTP moieties present in the segments of the templatestrands where the primers bind since this would be 10, 11 and 14respectively for the TS21, TS22 and TS13 primers. The results shown inFIG. 28 show that when carboxy-dUTP is used as a substrate,amplification can be carried out with only an 8° C. difference betweenthe denaturation and annealing/extension temperatures.

Example 10

Post Synthesis Modification of a Primer Extension Product

A primer extension reaction was carried out in the presence of allylamine dUTP. The template for the reaction had the following sequence:

5′-AGGTAACTTA AGATGGTCAG GCTGAAAGGA GGAACTATATC TGCAGAA-3′  (Sequence10)

The primer used for the reaction was TS 14 which has previously beendescribed. Reaction mixtures consisted of 1 ul of TS14 primer (100pmoles), 1 ul of template (100 pmoles), 2 ul of 25 mM MgCl₂, 2 ul of 400uM dGTP/dCTP/dATP, 2 ul allylamine ddUTP (ENZO Diagnostics, Farmingdale,N.Y.), 1 ul of Amplitaq DNA polymerase (Perkin-Elmer, Emeryville,Calif.), 1-3 ul of TAPS buffer and 0-2 ul of H₂O) for a final volume of12 ul for each reaction. TAPS buffer consisted of 200 mM TAPS (SIGMA,St. Louis, Mo.), 500 mM KCl with a ph of 9.6 unless otherwise indicated.Reactions had various levels of TAPS buffer and the pH was also varied.The particulars for each reaction are given inand amount of in thereactions were assessed and are described in FIGS. 29 and 30. As acontrol, reactions were also included that contained no enzyme or usedFluorescein ddUTP (ENZO Diagnostics, Farmingdale, N.Y.) instead of theallylamine ddUTP. Reaction mixes were heated to 94° C. for 1 minute andthen incubated at 68° C. for one hour.

Reactions were centrifuged briefly in a microfuge and 1 ul of 50 mMFluoroscein-5(6) carboxamido-caproic acid N-Hydroxysuccinimide ester(Fl-NHS ester) was added to each reaction and incubated at 37° C. forthree hours. Extent of synthesis and labeling were assessed byacrylamide gel electrophoresis. Fluorescent labeling was identified byputting the gel on a UV illuminator and taking a Polaroid picture usinga Wratten 58 Kodak Filter (SIGMA, St. Louis, Mo.). The gel was thenstained in Ethidium Bromide for 20 minutes followed by destaining for 20minutes and a picture taken using the normal filter. FIG. 29 shows thefluorescence provided by the incorporated fluoroscein (top gel) andEthidium Bromide staining (bottom gel) for each of the various reactionconditions. The photographs of these gels were also scanned and aphotonegative made of each of them. The results of this are shown inFIG. 30. The photonegative provides a better assessment of the resultsof the experiment. It can be seen in the upper photo that there is anextension product made that is capable of generating a fluorescentsignal under the various conditions used in the reactions. The highestlevel seems to have been achieved with 3×TAPS at pH 9.7. This experimentalso demonstrates that there is higher signal generation with thepost-synthesis modification than the control that used ddUTP that waspre-modified with fluorescein (lane 8). The extent of synthesis seen inthe lower gel also demonstrates the utility of this approach where itcan be seen by the presence of the upper band in lane 8 that there wasincorporation of the normal bases even though there was minimalincorporation of the pre-modified ddUTP.

Many obvious variations will be suggested to those of ordinary skill inthe art in light of the above detailed description of the presentinvention. All such obvious variations are fully contemplated andembraced by the scope and spirit of the present invention as set forthin the claims that now follow.

18 1 49 DNA Hepatitis B virus 1 catagcagca ggatgaagag gaatatgataggatgtgtct gcggcgttt 49 2 50 DNA Hepatitis B virus 2 tcctctaattccaggatcaa caacaaccag aggttttgca tggtcccgta 50 3 19 DNA Hepatitis Bvirus 3 ggatgtgtct gcggcgttt 19 4 20 DNA Hepatitis B virus 4 aggttttgcatggtcccgta 20 5 38 DNA Hepatitis B virus 5 ctcatcttct tattggttcttctggattat caaggtat 38 6 52 DNA Hepatitis B virus 6 catagcagcaggatgaagag gaatatgata gctggatgtg tctgcggcgt tt 52 7 53 DNA Hepatitis Bvirus 7 tcctctaatt ccaggatcaa caacaaccag tgcaggtttt gcatggtccc gta 53 860 DNA Hepatitis B virus 8 cctgctgcta tgcctcatct gacaaacggg caacatacctcctgctgcta tgcctcatct 60 9 23 DNA Escherichia coli 9 cgccagggtttcccagtcac gac 23 10 48 DNA Hepatitis B virus 10 aggtaactta agatggtcaggctgaaagga ggaactatat ctgcagaa 48 11 70 DNA Hepatitis B virus 11tgcgctgcta acaaagcccg aaaggaaggc tgaaaggagg aactatatgg cgtcatacga 60tatgaacgtt 70 12 70 DNA Hepatitis B virus 12 acgccaccat tgtttcgggctttccttccg actttcctcc ttgatatacg cgagtatgct 60 atacttgcaa 70 13 30 DNAHepatitis B virus 13 aatctagagc taacaaagcc cgaaaggaag 30 14 28 DNAHepatitis B virus 14 tgcgctgcta acaaagcccg aaaggaag 28 15 31 DNAHepatitis B virus 15 acccgcgctg ctaacaaagc ccgaaaggaa g 31 16 28 DNAHepatitis B virus 16 cgactttcct ccttgatata gacgtctt 28 17 27 DNAHepatitis B virus 17 cgactttcct ccttgatata cgcgagt 27 18 28 DNAHepatitis B virus 18 gatatacgcg agtatgctat acttgcaa 28

What is claimed is:
 1. A process for linearly amplifying a specificnucleic acid sequence comprising the steps of: 1) providing: saidspecific nucleic acid sequence, initial primers or nucleic acidconstructs comprising two segments, (A) a first segment (i) beingsufficiently complementary to a first portion of said specific nucleicacid sequence to bind or hybridize thereto and (ii) which provides fortemplate-dependent extension, and (B) a second segment being (i)substantially non-identical to said first segment, (ii) substantiallyidentical to a second portion of said specific nucleic acid sequence,(iii) which binds to a complementary sequence of said second segmentunder isostatic or limited cycling conditions after a firstcomplementary copy of said specific nucleic acid sequence has beenproduced by template dependent extension of said first segment, and (iv)thereby providing for subsequent binding of a first segment of anothersaid initial primer or nucleic acid construct to said first portion ofsaid specific nucleic acid sequence under isostatic or limited cyclingconditons, such that after primer extension, a second complementary copyis produced and displaces said first complementary copy; and substrates,buffer and a template-dependent polymerizing enzyme; and 2) incubatingsaid specific nucleic acid sequence and said initial primers or nucleicacid constructs in the presence of said substrates, buffer andtemplate-dependent polymerizing enzyme under isostatic or limitedcycling conditions; thereby linearly amplifying said specific nucleicacid sequence.
 2. The process of claim 1, wherein said first segment,said second segment or said first primer extension comprises at leastone modified nucleotide or nucleotide analog.
 3. The process of claim 2,wherein said second segment comprises at least one modified nucleotideor nucleotide analog which increases the thermodynamic stability of saidfirst segment to its complement in said primer extension.
 4. The processof claim 2 or 3, wherein said modified nucleotide or nucleotide analogcomprises an intercalating agent.
 5. The process of claim 1, whereinsaid first segment or said first primer extension or both comprises atleast one modified nucleotide or nucleotide analog.
 6. The process ofclaim 5, wherein said modified nucleotide or nucleotide analog decreasesthe thermodynamic stability of said first segment or said primerextension to its complement.
 7. The process of claim 6, wherein saidmodified nucleotide or nucleotide analog comprises a negatively chargedchemical group.
 8. The process of claim 7, wherein said negativelycharged chemical group comprises carboxylic acid.
 9. The process ofclaim 1, wherein said initial primer or nucleic acid construct comprisesa linear nucleic acid, a branched nucleic acid, an inverted nucleic acidor a peptide-nucleic acid.